U.S. patent application number 10/356442 was filed with the patent office on 2003-08-14 for compositions and methods for detecting protein modification and enzymatic activity.
Invention is credited to Cen, Hui, Shen, Li.
Application Number | 20030153014 10/356442 |
Document ID | / |
Family ID | 22568698 |
Filed Date | 2003-08-14 |
United States Patent
Application |
20030153014 |
Kind Code |
A1 |
Shen, Li ; et al. |
August 14, 2003 |
Compositions and methods for detecting protein modification and
enzymatic activity
Abstract
This invention relates generally to the field of protein
modification, e.g., post-translational modification. In particular,
the invention provides a method for detecting protein modification
profile in a sample, which method comprises: a) contacting a sample
containing or suspected of containing a target protein with a
capture molecule, or a plurality of capture molecules, immobilized
on a solid support, said capture molecule is capable of
specifically binding to said target protein, whereby said target
protein is immobilized on said solid support; and b) assessing
modification status and/or identity of said immobilized target
protein. Kits and arrays useful for detecting protein modification
are also provided. Arrays, kits and methods useful for detecting
enzymatic activities, especially protein modification enzymatic
activities, are further provided.
Inventors: |
Shen, Li; (Potomac, MD)
; Cen, Hui; (Oakland, CA) |
Correspondence
Address: |
Peng Chen
Morrison & Foerster LLP
Suite 500
3811 Valley Centre Drive
San Diego
CA
92130-2332
US
|
Family ID: |
22568698 |
Appl. No.: |
10/356442 |
Filed: |
January 30, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10356442 |
Jan 30, 2003 |
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09678644 |
Oct 3, 2000 |
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60158560 |
Oct 8, 1999 |
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Current U.S.
Class: |
435/7.9 |
Current CPC
Class: |
G01N 33/6842 20130101;
C12Q 1/485 20130101; G01N 33/6803 20130101 |
Class at
Publication: |
435/7.9 |
International
Class: |
G01N 033/53; G01N
033/542; G01N 033/537; G01N 033/543 |
Claims
1. A method for detecting protein modification of a target protein
in a sample, which method comprises: a) contacting a sample
containing or suspected of containing a target protein with a
capture molecule immobilized on a solid support, said capture
molecule is capable of specifically binding to said target protein,
whereby said target protein is immobilized on said solid support;
and b) assessing modification status and/or identity of said
immobilized target protein.
2. The method of claim 1, wherein a plurality of target proteins is
contacted with the immobilized capture molecule simultaneously.
3. The method of claim 2, wherein the plurality of target proteins
comprises a group of structurally and/or functionally related
proteins.
4. The method of claim 1, wherein a target protein is contacted
with a plurality of immobilized capture molecules
simultaneously.
5. The method of claim 1, wherein a plurality of target proteins is
contacted with a plurality of immobilized capture molecules
simultaneously.
6. The method of claim 1, wherein the capture molecule is capable
of specifically binding to both a modified and an unmodified forms
of a target protein.
7. The method of claim 1, wherein the capture molecule is capable
of specifically binding to a modified form of the target protein
but is not capable of specifically binding to an unmodified form of
the target protein.
8. The method of claim 1, wherein the capture molecule is an
antibody.
9. The method of claim 1, wherein the solid support is selected
from the group consisting of silicon, plastic, nylon, glass,
ceramic, photoresist, rubber or polymer support.
10. The method of claim 1, wherein the solid support comprises a
flat support, a set of sticks, or a set of beads.
11. The method of claim 10, wherein the flat support comprises a
slide, a chip, a filter, or a membrane.
12. The method of claim 1, wherein the protein modification is
selected from the group consisting of phosphorylation, acetylation,
methylation, ADP-ribosylation, addition of a polypeptide side
chain, addition of a hydrophobic group, and addition of a
carbohydrate.
13. The method of claim 12, wherein the phosphorylation is on an
amino acid residue selected from the group consisting of tyrosine,
serine, threonine and histidine.
14. The method of claim 12, wherein the addition of a polypeptide
side chain is the addition of ubiquitin.
15. The method of claim 12, wherein the addition of a hydrophobic
group is the addition of a fatty acid, addition of an isoprenoid,
or addition of a glycosyl-phosphatidyl inositol anchor.
16. The method of claim 15, wherein the fatty acid is myristate or
palmitate.
17. The method of claim 15, wherein the isoprenoid is famesyl or
genranylgenranyl.
18. The method of claim 12, wherein the carbohydrate group
comprises glycosyl.
19. The method of claim 6, wherein the modification status of the
immobilized target protein is assessed by contacting the
immobilized target protein with a detection molecule that is
capable of specifically binding to the modified target protein but
is not capable of specifically binding to the unmodified target
protein itself.
20. The method of claim 6, wherein the modification status of the
immobilized target protein is determined by a physical or chemical
means.
21. The method of claim 20, wherein the physical or chemical means
comprises chemical or radioisotopic label of the protein
modification moiety.
22. The method of claim 20, wherein the physical or chemical means
is selected from the group consisting of chromatographic,
electrophoretic, protein sequencing, mass spectrometry and NMR
means for detecting the protein modification moiety.
23. The method of claim 7, wherein the identity of the immobilized
target protein is assessed by contacting the immobilized target
protein with a detection molecule that is capable of specifically
binding to the unmodified target protein itself but is not capable
of specifically binding to the modified target protein.
24. The method of claim 1, wherein the sample is a biological
sample.
25. The method of claim 1, wherein the target protein is involved
in a biological pathway, belongs to a group of proteins with
identical or similar biological function, expressed in a stage of
cell cycle, expressed in a cell type, expressed in a tissue type,
expressed in an organ type, expressed in a developmental stage, a
protein whose expression and/or activity is altered in a disease or
disorder type or stage, or a protein whose expression and/or
activity is altered by drug or other treatments.
26. A method for identifying biologically distinguishable marker(s)
associated with a biosample, which comprises: 1) assessing protein
modification profile of a biosample through the method of claim 1;
2) assessing protein modification profile of a comparable control
biosample through the method of claim 1; and 3) comparing the
protein modification profile obtained in step 1) with the protein
modification profile obtained in step 2) to identify biologically
distinguishable protein modification profile marker(s) associated
with said biosample.
27. A kit for detecting protein modification, which kit comprises:
a) a capture molecule immobilized on a solid support, said capture
molecule is capable of specifically binding to a target protein;
and b) means for assessing modification status and/or identity of
said target protein.
28. The kit of claim 27, wherein a plurality of capture molecules
is immobilized on the solid support, each of said capture molecules
is capable of specifically binding to a member protein of a group
of structurally and/or functionally related target proteins.
29. The kit of claim 27, wherein the capture molecule is capable of
specifically binding to both a modified and an unmodified forms of
a target protein.
30. The kit of claim 27, wherein the capture molecule is capable of
specifically binding to a modified form of the target protein but
is not capable of specifically binding to an unmodified form of the
target protein.
31. The kit of claim 27, wherein the capture molecule is an
antibody.
32. The kit of claim 27, wherein the protein modification is
selected from the group consisting of phosphorylation, acetylation,
methylation, ADP-ribosylation, addition of a polypeptide side
chain, addition of a hydrophobic group, and addition of a
carbohydrate.
33. The kit of claim 29, wherein the modification status of the
immobilized target protein is assessed by contacting the
immobilized target protein with a detection molecule that is
capable of specifically binding to the modified target protein but
is not capable of specifically binding to the unmodified target
protein itself.
34. The kit of claim 29, wherein the modification status of the
immobilized target protein is determined by a physical or chemical
means.
35. The kit of claim 30, wherein the identity of the immobilized
target protein is assessed by contacting the immobilized target
protein with a detection molecule that is capable of specifically
binding to the unmodified target protein itself but is not capable
of specifically binding to the modified target protein.
36. The kit of claim 27, further comprising: a) instructions for
using the kit; b) reagents and buffers; and/or c) a container(s)
for the kit contents.
37. An array of protein capture molecules, which array comprises:
a) a solid support; and b) a plurality of capture molecules
immobilized on said solid support, wherein each of said molecules
is capable of specifically binding to both a modified and an
unmodified form of a member protein of a plurality of target
proteins.
38. The array of claim 37, wherein the plurality of target proteins
comprises a group of structurally and/or functionally related
proteins.
39. The array of claim 38, wherein the modified and unmodified
forms of the same target protein have different biological
activities.
40. The array of claim 38, wherein the modified and unmodified
forms of the same target protein represent different physiological
conditions or biological statuses.
41. The array of claim 40, wherein the array is used to identify
pathway activation.
42. The array of claim 40, wherein the array is used to identify
activation of a group of structurally and/or functionally related
protein.
43. The array of claim 40, wherein the array is used to generate a
modification profile correlated to a physiological condition, drug
treatment and disease.
44. The array of claim 40, wherein the array is used to identify a
physiological or pathological status.
45. The array of claim 40, wherein the array is used to record
biological perturbation caused by drug and other treatment.
46. An array of protein capture molecules, which array comprises:
a) a solid support; and b) a plurality of capture molecules
immobilized on said solid support, wherein each of said molecules
is capable of specifically binding to an epitope generated by a
specific modification moiety of a modified protein.
47. The array of claim 46, wherein the modified protein is Rb.
48. An array of enzyme substrates, which array comprises: a) a
solid support; and b) a plurality of substrates immobilized on said
solid support, wherein each of said substrates is a substrate of a
member enzyme of a group of structurally and/or functionally
related enzymes.
49. The array of claim 48, wherein at least one of the member
enzymes catalyzes a protein modification reaction.
50. A kit for detecting enzymatic activity, which kit comprises: a)
the array of claim 48; and b) means for assessing activity of each
of the member enzymes.
51. A method for detecting enzymatic activity in a sample, which
method comprises: a) contacting a sample containing or suspected of
containing a group of structurally and/or functionally related
target enzymes with a plurality of substrates immobilized on a
solid support, wherein each of said substrates is a substrate of a
member enzyme of said group of target enzymes under conditions
suitable for said target enzymes to catalyze enzymatic reactions
involving said immobilized substrates; and b) assessing enzymatic
activities of said target enzymes.
52. The method of claim 51, wherein at least one of the target
enzymes catalyzes a protein modification reaction.
Description
[0001] This application claims the benefit of the priority date of
the U.S. Provisional Patent Application Serial No. 60/158,560,
filed Oct. 8, 1999 under 35 U.S.C. .sctn.119(e). The content of the
above-referenced application is herein incorporated by reference in
its entirety.
TECHNICAL FIELD
[0002] This invention relates generally to the field of protein
modification, e.g., post-translational modification. In particular,
the invention provides a method for detecting protein modification
profile in a sample, which method comprises: a) contacting a sample
containing or suspected of containing a target protein with a
capture molecule, or a plurality of capture molecules, immobilized
on a solid support, said capture molecule is capable of
specifically binding to said target protein, whereby said target
protein is immobilized on said solid support; and b) assessing
modification status and/or identity of said immobilized target
protein. Kits and arrays useful for detecting protein modification
are also provided. Arrays, kits and methods useful for detecting
enzymatic activities, especially protein modification enzymatic
activities, are further provided.
BACKGROUND ART
[0003] Westernblot and immunoprecipitation (IP) are the most
frequently used procedures in laboratory research lab for analyzing
a protein for its expression, molecular weight, degradation, and
conformational changes. Westernblot procedures detect the presence
of an antigen of interest by an antigen-specific antibody after
separating proteins by electrophoresis on an acrylamide gel,
transferring separated proteins from the acrylamide gel to a
nitrocellulose membrane and immunoblotting. IP is a procedure that
is used to study the properties of a specific molecule by
immunoprecipitating the molecule from a protein mixture and
separating the mixture of immunoprecipitants by electrophoresis.
Despite the popular use of Westernblot and IP, these procedures
remain time-consuming (requiring a minimum of two days to complete
each) and are complicated. Both methods rely on electrophoresis for
protein separation, and thus each Westernblot and IP procedure is
optimally used for analyzing a single protein or at best a few
proteins having different molecular weights. To analyze a few
proteins with similar molecular weights, separate Westernblots with
antibodies specific to each of the proteins of interest need to be
performed, requiring a copy of the identical antigen-bearing
nitrocellulose membrane for performing each blot for each antigen.
A procedure combining IP with Westernblot may be used to analyze
proteins with similar molecular weights for their
post-translational modification such as tyrosine phosphorylation
using anti-phosphotyrosine antibody. However, each antigen of
interest requires its own blot.
[0004] A kinase assay can be performed by mixing a substrate of the
kinase enzyme with enzyme containing solution for a period of time
in the presence of r-labeled ATP followed by electrophoresis to
separate the enzyme substrate from the rest of proteins in the
mixture. The enzyme activity is reflected by the amount of
radioactivity incorporated into the substrate.
[0005] ELISA assays are widely used for screening agonists and
antagonists for a particular protein, a particular
post-translational modification of a protein and a biological
activity of a protein. Recently, several ELISA assays have been
developed for measuring post-translational modification such as
tyrosine phosphorylation. These include an ELISA assay for
Heregulin-induced ErbB2 phosphorylation by Sadick et al. Analytical
Chemistry, 235:207-214 (1996) and an ELISA assay for VEGF-induced
Flk-1/KDR phosphorylation. Many groups also have developed ELISA
assay for a specific kinase. For the ELISA assay, the cell lysate
from each sample is added into a 96-well plate pre-coated with an
antibody against a desired antigen to allow binding of the desired
protein onto the surface of 96-well. After the binding, cell lysate
is removed from the well and replaced with another antigen-specific
antibody that is directly or indirectly conjugated with enzyme. The
amount of the antigen of interest is then determined by the
activity of the enzyme activity. ELISA assays are designed to
screen a large number of samples against a single antigen rather
than analyze multiple antigens on a single 96-well plate.
[0006] Two-dimensional gel electrophoresis is widely used to
analyze genome-wide protein expression and modification. However,
the accurate determination of each protein remains difficult since
the resolution of the current two-dimensional gel electrophoresis
(3000-5000 spots) is far below the total number of cellular
proteins. Some protein array technologies have also been developed
for gene expression and antibody screening. For these technologies,
a library of proteins is immobilized on a two-dimensional array.
These proteins are either individually prepared and spotted on a
specific area of the array or derived from a nucleic acid array
through in situ translation. These antigen arrays are used to
screen antibodies, ligands and receptors that interact with the
antigen on the arrays.
[0007] Recently, efforts have been made to develop antibody
microarrays for simultaneous detection of protein expression in
clinical analytes such as microbiles and immunoglobulins in the
blood. These detection systems focus on the detection of a
particular protein expression. A ligand binding mass-sensing assay
for quantifying a ligand based on its specific affinity for a
chemically modified solid material is described in Silzel et al,
Clinical Chem. 44:92036-2043 (1998). The assays provide
laser-induced fluorescence detection and can detect microscopic
volumes of several different capture reagents. See also, Rowe et
al, Anal Chem 71,433-439 (1999) which describes a
fluorescence-based immunosensor for simultaneous analysis and
detection of clinical analytes: A pattern array of recognition
elements is immobilized on the surface of a planar waveguide used
to capture analyte present in samples; bound analyte is then
quantified by fluorescent-detector molecules.
[0008] Therefore, it is an object of the present invention to
provide compositions, e.g., arrays and kits, and methods for
detecting protein or peptide modification and enzymatic
activity.
DISCLOSURE OF THE INVENTION
[0009] The methods, kits, arrays and other compositions of the
invention provide for contacting a biologically active or activated
sample of proteins with a solid support array of capture molecules
specific for target proteins that may or may not be present in the
sample. The post-translational modified proteins bound to the
capture molecules on the solid support are detected using detection
means that specifically recognize the modification moiety. The
detection means herein is contacting immobilized target proteins
(immobilized by binding with capture molecules that immobilized on
the solid support) to a detection molecule that specifically bind
to modification moiety, a detection molecule that specifically bind
to target protein, or contacting immobilized target proteins to a
detection reaction that specifically react with a modification
moiety, or physical means that specifically recognize a residue
present in the modification moiety. The invention provides a means
for detecting protein modifications in order to deduce what
biological activity or activities, or biological status are present
in a given sample of proteins tested. A major advantage of the
invention is that the analysis can be simultaneous for whole groups
of proteins in a given sample by analyzing either a single type of
protein modification at a time, or a profile of protein
modification that indicates some information about the biological
activity or status present in the sample. The method is especially
useful for generating a profile of protein modification. The
principles of the invention are also applied to detecting enzymatic
activity in an enzymatically active or activated sample of
proteins. The invention can be used for making comparisons between
control samples and samples that represent a changed condition. The
condition can include for example, disease, disease progression, a
disease stage, a developmental stage, drug treatment, chemical
treatment, physical change, biological change, different tissues,
different animals, different cells, different dosages and any other
condition it would be useful to identify and characterize by a
comparison of protein modification or enzymatic activity.
[0010] Method
[0011] In one aspect, the present invention is directed to a method
for detecting protein modification of a target protein in a sample,
which method comprises: a) contacting a sample containing or
suspected of containing a target protein with a capture molecule
immobilized on a solid support, said capture molecule is capable of
specifically binding to said target protein, whereby said target
protein is immobilized on said solid support; and b) assessing
modification status and/or identity of said immobilized target
protein.
[0012] Although the present method can be used to assess the
protein modification status of a single target protein at a time,
the present method is preferably used in a high-throughput format.
For example, the protein modification status (or profile) of a
plurality of target proteins can be assessed simultaneously by
contacting the plurality of target proteins with an immobilized
capture molecule, or a plurality of immobilized capture molecules
simultaneously. Alternatively, the protein modification status (or
profile) of a single target protein can be assessed simultaneously
by contacting a target protein with a plurality of immobilized
capture molecules simultaneously. Preferably, the protein
modification status (or profile) of a plurality of target proteins
can be assessed simultaneously by contacting the plurality of
target proteins with a plurality of immobilized capture molecules
simultaneously.
[0013] The protein modification status (or profile) of any
plurality, i.e., group, of target proteins can be assessed by the
present method. Preferably, the protein modification status (or
profile) of a group of structurally and/or functionally related
proteins are assessed.
[0014] Any molecule, or complex or combination therefor, that is
capable of specifically binding to a target protein, or one or more
member(s) of a plurality of target proteins, can be used as the
capture molecule in the present method. In one specific embodiment,
the capture molecule is capable of specifically binding to both a
modified and an unmodified forms of a target protein. In another
specific embodiment, the capture molecule is capable of
specifically binding to a modified form of the target protein but
is not capable of specifically binding to an unmodified form of the
target protein. In a preferred embodiment, the capture molecule is
an antibody, e.g., a polyclonal antibody, a monoclonal antibody, an
antibody fragment retaining its desired binding specificity, or a
combination thereof.
[0015] Any suitable solid support can be used in the present
method. In one example, the solid support can be a silicon,
plastic, nylon, glass, ceramic, photoresist, rubber or polymer
support. The solid support can be in any kind of suitable geometric
forms, e.g., a flat support, a set of sticks, or a set of beads.
Exemplary flat supports can comprise a slide, a chip, a filter, or
a membrane.
[0016] Any protein modification, especially post-translational
protein modification, can be assessed by the present method.
Exemplary protein modifications that can be assessed by the present
method include phosphorylation, acetylation, methylation,
ADP-ribosylation, addition of a polypeptide side chain, addition of
a hydrophobic group, and addition of a carbohydrate. In one
specific embodiment, the phosphorylation to be assessed is
phosphorylation on tyrosine, serine, threonine or histidine
residue. In another specific embodiment, the addition of a
polypeptide side chain to be assessed is the addition of ubiquitin.
In still another specific embodiment, the addition of a hydrophobic
group to be assessed is the addition of a fatty acid, e.g.,
myristate or palmitate, addition of an isoprenoid, e.g., farnesyl
or genranylgenranyl, or addition of a glycosyl-phosphatidyl
inositol anchor, e.g., a carbohydrate group comprises glycosyl.
[0017] The present method can be used in different formats. For
example, the target protein can be immobilized via the specific
interaction between a capture molecule and the peptidic portion of
the target protein, and then the protein modification status of the
immobilized target protein is assessed. Alternatively, the target
protein can be immobilized via the specific interaction between a
capture molecule and the modification moiety or the combination of
the modification moiety and the peptidic portion of the target
protein, and then the identity of the immobilized target protein is
assessed. Accordingly, in one specific embodiment, the target
protein is first immobilized by a capture molecule that is capable
of specifically binding to both a modified and an unmodified forms
of a target protein, and then the modification status of the
immobilized target protein is assessed by contacting the
immobilized target protein with a detection molecule that is
capable of specifically binding to the modified target protein but
is not capable of specifically binding to the unmodified target
protein itself. In this embodiment, the modification status of the
immobilized target protein can also be determined by other suitable
physical or chemical means. For example, the physical or chemical
means can comprise chemical or radioisotopic label of the protein
modification moiety. Alternatively, the physical or chemical means
can comprise any suitable analytical means, e.g., chromatographic,
electrophoretic, protein sequencing, mass spectrometry and NMR
means, for detecting the protein modification moiety. In another
specific embodiment, the target protein is first immobilized by a
capture molecule that is capable of specifically binding to a
modified form of the target protein but is not capable of
specifically binding to an unmodified form of the target protein,
and then the identity of the immobilized target protein is assessed
by contacting the immobilized target protein with a detection
molecule that is capable of specifically binding to the unmodified
target protein itself but is not capable of specifically binding to
the modified target protein.
[0018] The protein modification status of a target protein, or a
plurality of target proteins, in any sample can be assessed by the
present method. Preferably, the sample to be assessed is a
biological sample.
[0019] The protein modification status of any target protein, or
any plurality of target proteins, can be assessed by the present
method. Preferably, the target protein to be assessed is involved
in a biological pathway, belongs to a group of proteins with
identical or similar biological function, expressed in a stage of
cell cycle, expressed in a cell type, expressed in a tissue type,
expressed in an organ type, expressed in a developmental stage, a
protein whose expression and/or activity is altered in a disease or
disorder type or stage, or a protein whose expression and/or
activity is altered by drug or other treatments.
[0020] In another aspect, the present invention is directed to a
method for identifying biologically distinguishable marker(s)
associated with a biosample, which comprises: 1) assessing protein
modification profile of a biosample through the above-described
method; 2) assessing protein modification profile of a comparable
control biosample through the above-described method; and 3)
comparing the protein modification profile obtained in step 1) with
the protein modification profile obtained in step 2) to identify
biologically distinguishable protein modification profile marker(s)
associated with said biosample. Preferably, the identified
biologically distinguishable protein modification profile marker(s)
are indicative of the protein modification profile of the
biological source from which the biosample is derived.
[0021] Kits and Arrays
[0022] In still another aspect, the present invention is directed
to a kit for detecting protein modification, which kit comprises:
a) a capture molecule immobilized on a solid support, said capture
molecule is capable of specifically binding to a target protein;
and b) means for assessing modification status and/or identity of
said target protein.
[0023] In yet another aspect, the present invention is directed to
an array of protein capture molecules, which array comprises: a) a
solid support; and b) a plurality of capture molecules immobilized
on said solid support, wherein each of said molecules is capable of
specifically binding to both a modified and an unmodified form of a
member protein of a plurality of target proteins. Preferably, the
plurality of target proteins comprises a group of structurally
and/or functionally related proteins.
[0024] In yet another aspect, the present invention is directed to
an array of protein capture molecules, which array comprises: a) a
solid support; and b) a plurality of capture molecules immobilized
on said solid support, wherein each of said molecules is capable of
binding to a specific epitope with a modification moiety of a
modified protein, e.g., Rb.
[0025] Array, Kits and Methods for Detecting Enzymatic Activity
[0026] In yet another aspect, the present invention is directed to
an array of enzyme substrates, which array comprises: a) a solid
support; and b) a plurality of substrates immobilized on said solid
support, wherein each of said substrates is a substrate of a member
enzyme of a group of structurally and/or functionally related
enzymes. Preferably, at least one of the member enzymes catalyzes a
protein modification reaction.
[0027] In yet another aspect, the present invention is directed to
a kit for detecting enzymatic activity, which kit comprises: a) an
array comprising a solid support, and a plurality of substrates
immobilized on said solid support, wherein each of said substrates
is a substrate of a member enzyme of a group of structurally and/or
functionally related enzymes; and b) means for assessing activity
of each of the member enzymes.
[0028] In yet another aspect, the present invention is directed to
a method for detecting enzymatic activity in a sample, which method
comprises: a) contacting a sample containing or suspected of
containing a group of structurally and/or functionally related
target enzymes with a plurality of substrates immobilized on a
solid support, wherein each of said substrates is a substrate of a
member enzyme of said group of target enzymes under conditions
suitable for said target enzymes to catalyze enzymatic reactions
involving said immobilized substrates; and b) assessing enzymatic
activities of said target enzymes. Preferably, at least one of the
target enzymes catalyzes a protein modification reaction.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 illustrates the principle of the P-Y ProArray. Step
1: Specific capturing antibodies against PTKs are immobilized on a
two-dimensional surface. Step 2: PTKs from the cell lysate are
captured on the two-dimensional surface at specific positions. Step
3: An enzyme- or a fluorescent-dye-conjugated anti-P-Y antibody
binds to P-Y residues in the PTK. Step 4: A chemiluminescent or
fluorescent signal is generated to indicate P-Y levels in the
PTKs.
[0030] FIG. 2 illustrates the arrangement of an exemplary membrane
protein array.
MODES OF CARRYING OUT THE INVENTION
[0031] A. Definitions
[0032] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which this invention belongs. All
patents, applications, published applications and other
publications and sequences from GenBank and other databases
referred to herein are incorporated by reference in their entirety.
If a definition set forth in this section is contrary to or
otherwise inconsistent with a definition set forth in applications,
published applications and other publications and sequences from
GenBank and other data bases that are herein incorporated by
reference, the definition set forth in this section prevails over
the definition that is incorporated herein by reference.
[0033] As used herein, "a" or "an" means "at least one" or "one or
more."
[0034] As used herein, "protein" encompasses polypeptide,
oligopeptide and peptide.
[0035] As used herein, "protein modification" refers to addition of
a peptidic or non-peptidic moiety to a protein that cannot be
considered as the elongation of the peptidic chain of the protein.
The addition of the peptidic or non-peptidic moiety can be in vivo
or in vitro. The peptidic or non-peptidic moiety can be added to a
pure protein or a protein or peptidic component of a complex
containing such protein or peptide. Preferably, "protein
modification" refers to post-translational protein modification.
Exemplary post-translational protein modification include
phosphorylation, acetylation, methylation, ADP-ribosylation,
addition of a polypeptide side chain, addition of a hydrophobic
group, and addition of a carbohydrate.
[0036] As used herein, "capture molecule" refers to a molecule, or
complex or combination therefor, that is capable of specifically
binding to a target protein, or one or more member(s) of a
plurality of target proteins. The capture molecule can be peptides,
proteins, e.g., antibodies or receptors, oligonucleotides, nucleic
acids, e.g., protein-binding DNA or RNA molecules, vitamins,
oligosaccharides, carbohydrates, lipids, small molecules, or a
complex thereof.
[0037] As used herein, "macromolecule" refers to a molecule that,
without attaching to another molecule, is capable of generating an
antibody that specifically binds to the macromolecule.
[0038] As used herein, "small molecule" refers to a molecule that,
without forming homo-aggregates or without attaching to a
macromolecule or adjuvant, is incapable of generating an antibody
that specifically binds to the small molecule. Preferably, the
small molecule has a molecular weight that is about or less than
10,000 daltons. More preferably, the small molecule has a molecular
weight that is about or less than 5,000 dalton.
[0039] As used herein, "vitamin" refers to a trace organic
substance required in certain biological species. Most vitamins
function as components of certain coenzymes.
[0040] As used herein, "lipid" refers to water-insoluble, oily or
greasy organic substances that are extractable from cells and
tissues by nonpolar solvents, such as chloroform or ether.
[0041] As used herein, a "receptor" refers to a molecule that has
an affinity for a given ligand. Receptors may be
naturally-occurring or synthetic molecules. Receptors may also be
referred to in the art as anti-ligands. As used herein, the
receptor and anti-ligand are interchangeable. Receptors can be used
in their unaltered state or as aggregates with other species.
Receptors may be attached, covalently or noncovalently, or in
physical contact with, to a binding member, either directly or
indirectly via a specific binding substance or linker. Examples of
receptors, include, but are not limited to: antibodies, cell
membrane receptors surface receptors and internalizing receptors,
monoclonal antibodies and antisera reactive with specific antigenic
determinants [such as on viruses, cells, or other materials],
drugs, polynucleotides, nucleic acids, peptides, cofactors,
lectins, sugars, polysaccharides, cells, cellular membranes, and
organelles.
[0042] Examples of receptors and applications using such receptors,
include but are not restricted to:
[0043] a) enzymes: specific transport proteins or enzymes essential
to survival of microorganisms, which could serve as targets for
antibiotic [ligand] selection;
[0044] b) antibodies: identification of a ligand-binding site on
the antibody molecule that combines with the epitope of an antigen
of interest may be investigated; determination of a sequence that
mimics an antigenic epitope may lead to the development of vaccines
of which the immunogen is based on one or more of such sequences or
lead to the development of related diagnostic agents or compounds
useful in therapeutic treatments such as for auto-immune
diseases
[0045] c) nucleic acids: identification of ligand, such as protein
or RNA, binding sites;
[0046] d) catalytic polypeptides: polymers, preferably
polypeptides, that are capable of promoting a chemical reaction
involving the conversion of one or more reactants to one or more
products; such polypeptides generally include a binding site
specific for at least one reactant or reaction intermediate and an
active functionality proximate to the binding site, in which the
functionality is capable of chemically modifying the bound reactant
[see, e.g., U.S. Pat. No. 5,215,899];
[0047] e) hormone receptors: determination of the ligands that bind
with high affinity to a receptor is useful in the development of
hormone replacement therapies; for example, identification of
ligands that bind to such receptors may lead to the development of
drugs to control blood pressure; and
[0048] f) opiate receptors: determination of ligands that bind to
the opiate receptors in the brain is useful in the development of
less-addictive replacements for morphine and related drugs.
[0049] As used herein, "antibody" includes antibody fragments, such
as Fab fragments, which are composed of a light chain and the
variable region of a heavy chain.
[0050] As used herein, "humanized antibodies" refer to antibodies
that are modified to include "human" sequences of amino acids so
that administration to a human will not provoke an immune response.
Methods for preparation of such antibodies are known. For example,
the hybridoma that expresses the monoclonal antibody is altered by
recombinant DNA techniques to express an antibody in which the
amino acid composition of the non-variable regions is based on
human antibodies. Computer programs have been designed to identify
such regions.
[0051] As used herein the term "assessing" is intended to include
quantitative and qualitative determination in the sense of
obtaining an absolute value for the amount or concentration of the
analyte, e.g., a homocysteine co-substrate, present in the sample,
and also of obtaining an index, ratio, percentage, visual or other
value indicative of the level of analyte in the sample. Assessment
may be direct or indirect and the chemical species actually
detected need not of course be the analyte itself but may for
example be a derivative thereof or some further substance.
[0052] As used herein, "a group of structurally and/or functionally
related proteins" refers to a group of proteins, at their natural
status, that are structurally linked, located at the same cellular
locations, e.g., cellular organelles, located in the same tissues
or organs, expressed and/or be functional in the same biological
stages, e.g., a particular cell cycle stage or developmental stage,
or expressed and/or be functional in the same biological pathway,
e.g., a particular metabolism pathway, signal transduction pathway,
etc. The "group of structurally and/or functionally related
proteins" need only include at least two proteins belonging to the
same group. The "group of structurally and/or functionally related
proteins" can preferably include more than two proteins belonging
to the same group, e.g., a majority of or even all the proteins
belonging to the same group.
[0053] As used herein, "nutrient or storage protein" refers to a
protein that is used by the cell as the nutrient source or storage
form for such nutrient. Non-limiting examples of nutrient or
storage proteins include gliadin, ovalbumin, casein, and
ferritin.
[0054] As used herein, "contractile or motile protein" refers to a
protein that endows cells and organisms with the ability to
contract, to change shape, or to move about. Non-limiting examples
of contractile or motile proteins include actin, myosin, tubulin
and dynein.
[0055] As used herein, "structural protein" refers to a protein
that serves as supporting filaments, cables, or sheets to give
biological structures strength or protection. Non-limiting examples
of structural proteins include keratin, fibroin, collagen, elastin
and proteoglycans.
[0056] As used herein, "defense protein" refers to a protein that
defends organisms against invasion by other species or protect them
from injury. Non-limiting examples of defense proteins include
antibodies, fibrinogen, thrombin, botulinus toxin, diphtheria
toxin, snake venoms and ricin.
[0057] As used herein, "regulatory protein" refers to a protein
that helps regulate cellular or physiological activity.
Non-limiting examples of regulatory proteins include insulin,
growth hormones, corticotropin and repressors.
[0058] As used herein, "the capture molecule is capable of
specifically binding to both a modified and an unmodified forms of
a target protein" means that the capture molecule can bind to the
target protein through specific interaction between the capture
molecule and the peptidic portion of the target protein, and cannot
bind to the target protein through specific interaction between the
capture molecule and the modification moiety, or the combination of
the peptidic portion and the modification moiety of the target
protein.
[0059] As used herein, "the capture molecule is capable of
specifically binding to a modified form of the target protein but
is not capable of specifically binding to an unmodified form of the
target protein" means that the capture molecule can bind to the
target protein through specific interaction between the capture
molecule and the modification moiety, or the combination of the
peptidic portion and the modification moiety of the target protein,
and cannot bind to the target protein through specific interaction
between the capture molecule and the peptidic portion of the target
protein alone.
[0060] As used herein, "sample" refers to anything which may
contain an analyte for which an analyte assay is desired. The
sample may be a biological sample, such as a biological fluid or a
biological tissue. Examples of biological fluids include urine,
blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal
fluid, tears, mucus, amniotic fluid or the like. Biological tissues
are aggregates of cells, usually of a particular kind together with
their intercellular substance that form one of the structural
materials of a human, animal, plant, bacterial, fungal or viral
structure, including connective, epithelium, muscle and nerve
tissues. Examples of biological tissues also include organs,
tumors, lymph nodes, arteries and individual cell(s). The sample
may also be a mixture of target protein containing molecules
prepared in vitro.
[0061] As used herein, "a comparable control biosample" refers to a
control biosample that is only different in one or more defined
aspects from the biosample, and the present methods, kits or arrays
are used to identify the effects, if any, of these defined
difference(s) between the biosample to be assessed and the control
biosample on the protein modification profile of the biosample. For
example, the biosample to be assessed and the control biosample can
be derived from physiological normal conditions and comparable
physiological abnormal conditions, can be subjected to different
physical, chemical, physiological or drug treatments, or can be
derived from different biological stages, etc.
[0062] As used herein, "a group of structurally and/or functionally
related enzymes" refers to a group of enzymes, at their natural
status, that are structurally linked, located at the same cellular
locations, e.g., cellular organelles, located in the same tissues
or organs, expressed and/or be functional in the same biological
stages, e.g., a particular cell cycle stage or developmental stage,
or expressed and/or be functional in the same biological pathway,
e.g., a particular metabolism pathway, signal transduction pathway,
or act as a regulator for a pathway activation or a biological
function, etc. The "group of structurally and/or functionally
related enzymes" need only include at least two enzymes belonging
to the same group. The "group of structurally and/or functionally
related enzymes" can preferably include more than two enzymes
belonging to the same group, e.g., a majority of or even all the
enzymes belonging to the same group.
[0063] As used herein, "expressed in a tissue or organ specific
manner" refers to a gene expression pattern in which a gene is
expressed, either transiently or constitutively, only in certain
tissues or organs, but not in other tissues or organs.
[0064] As used herein, "tissue" refers to a collection of similar
cells and the intracellular substances surrounding them. There are
four basic tissues in the body: 1) epithelium; 2) connective
tissues, including blood, bone, and cartilage; 3) muscle tissue;
and 4) nerve tissue.
[0065] As used herein, "organ" refers to any part of the body
exercising a specific function, as of respiration, secretion or
digestion.
[0066] As used herein, "plant" refers to any of various
photosynthetic, eucaryotic multi-cellular organisms of the kingdom
Plantae, characteristically producing embryos, containing
chloroplasts, having cellulose cell walls and lacking
locomotion.
[0067] As used herein, "animal" refers to a multi-cellular organism
of the kingdom of Animalia, characterized by a capacity for
locomotion, nonphotosynthetic metabolism, pronounced response to
stimuli, restricted growth and fixed bodily structure. Non-limiting
examples of animals include birds such as chickens, vertebrates
such fish and mammals such as mice, rats, rabbits, cats, dogs,
pigs, cows, ox, sheep, goats, horses, monkeys and other non-human
primates.
[0068] As used herein, "bacteria" refers to small prokaryotic
organisms (linear dimensions of around 1 .mu.m) with
non-compartmentalized circular DNA and ribosomes of about 70S.
Bacteria protein synthesis differs from that of eukaryotes. Many
anti-bacterial antibiotics interfere with bacteria proteins
synthesis but do not affect the infected host.
[0069] As used herein, "eubacteria" refers to a major subdivision
of the bacteria except the archaebacteria. Most Gram-positive
bacteria, cyanobacteria, mycoplasmas, enterobacteria, pseudomonas
and chloroplasts are eubacteria. The cytoplasmic membrane of
eubacteria contains ester-linked lipids; there is peptidoglycan in
the cell wall (if present); and no introns have been discovered in
eubacteria.
[0070] As used herein, "archaebacteria" refers to a major
subdivision of the bacteria except the eubacteria. There are three
main orders of archaebacteria: extreme halophiles, methanogens and
sulphur-dependent extreme thermophiles. Archaebacteria differs from
eubacteria in ribosomal structure, the possession (in some case) of
introns, and other features including membrane composition.
[0071] As used herein, "virus" refers to an obligate intracellular
parasite of living but non-cellular nature, consisting of DNA or
RNA and a protein coat. Viruses range in diameter from about 20 to
about 300 nm. Class I viruses (Baltimore classification) have a
double-stranded DNA as their genome; Class II viruses have a
single-stranded DNA as their genome; Class III viruses have a
double-stranded RNA as their genome; Class IV viruses have a
positive single-stranded RNA as their genome, the genome itself
acting as mRNA; Class V viruses have a negative single-stranded RNA
as their genome used as a template for mRNA synthesis; and Class VI
viruses have a positive single-stranded RNA genome but with a DNA
intermediate not only in replication but also in mRNA synthesis.
The majority of viruses are recognized by the diseases they cause
in plants, animals and prokaryotes. Viruses of prokaryotes are
known as bacteriophages.
[0072] As used herein, "fungus" refers to a division of eucaryotic
organisms that grow in irregular masses, without roots, stems, or
leaves, and are devoid of chlorophyll or other pigments capable of
photosynthesis. Each organism (thallus) is unicellular to
filamentous, and possesses branched somatic structures (hyphae)
surrounded by cell walls containing glucan or chitin or both, and
containing true nuclei.
[0073] As used herein, "disease or disorder" refers to a
pathological condition in an organism resulting from, e.g.,
infection or genetic defect, and characterized by identifiable
symptoms.
[0074] As used herein, "infection" refers to invasion of the body
of a multi-cellular organism with organisms that have the potential
to cause disease.
[0075] As used herein, "infectious organism" refers to an organism
that is capable to cause infection of a multi-cellular organism.
Most infectious organisms are microorganisms such as viruses,
bacteria and fungi.
[0076] As used herein, neoplasm (neoplasia) refers to abnormal new
growth, and thus means the same as tumor, which may be benign or
malignant. Unlike hyperplasia, neoplastic proliferation persists
even in the absence of the original stimulus.
[0077] As used herein, cancer refers to a general term for diseases
caused by any type of malignant tumor.
[0078] As used herein, "an immune system disease or disorder"
refers to a pathological condition caused by a defect in the immune
system. The immune system is a complex and highly developed system,
yet its mission is simple: to seek and kill invaders. If a person
is born with a severely defective immune system, death from
infection by a virus, bacterium, fungus or parasite will occur. In
severe combined immunodeficiency, lack of an enzyme means that
toxic waste builds up inside immune system cells, killing them and
thus devastating the immune system. A lack of immune system cells
is also the basis for DiGeorge syndrome: improper development of
the thymus gland means that T cell production is diminished. Most
other immune disorders result from either an excessive immune
response or an `autoimmune attack`. For example, asthma, familial
Mediterranean fever and Crohn disease (inflammatory bowel disease)
all result from an over-reaction of the immune system, while
autoimmune polyglandular syndrome and some facets of diabetes are
due to the immune system attacking self cells and molecules. A key
part of the immune system's role is to differentiate between
invaders and the body's own cells--when it fails to make this
distinction, a reaction against self cells and molecules causes
autoimmune disease.
[0079] As used herein, "a metabolism disease or disorder" refers to
a pathological condition caused by errors in metabolic processes.
Metabolism is the means by which the body derives energy and
synthesizes the other molecules it needs from the fats,
carbohydrates and proteins we eat as food, by enzymatic reactions
helped by minerals and vitamins. There is a significant level of
tolerance of errors in the system: often, a mutation in one enzyme
does not mean that the individual will suffer from a disease. A
number of different enzymes may compete to modify the same
molecule, and there may be more than one way to achieve the same
end result for a variety of metabolic intermediates. Disease will
only occur if a critical enzyme is disabled, or if a control
mechanism for a metabolic pathway is affected.
[0080] As used herein, "a muscle and bone disease or disorder"
refers to a pathological condition caused by defects in genes
important for the formation and function of muscles, and connective
tissues. Connective tissue is used herein as a broad term that
includes bones, cartilage and tendons. For example, defects in
fibrillin--a connective tissue protein that is important in making
the tissue strong yet flexible--cause Marfan syndrome, while
diastrophic dysplasia is caused by a defect in a sulfate
transporter found in cartilage. Two diseases that originate through
a defect in the muscle cells themselves are Duchenne muscular
dystrophy (DMD) and myotonic dystrophy (DM). DM is another `dynamic
mutation` disease, similar to Huntington disease, that involves the
expansion of a nucleotide repeat, this time in a muscle protein
kinase gene. DMD involves a defect in the cytoskeletal protein,
dystrophin, which is important for maintaining cell structure.
[0081] As used herein, "a nervous system disease or disorder"
refers to a pathological condition caused by defects in the nervous
system including the central nervous system, i.e., brain, and the
peripheral nervous system. The brain and nervous system form an
intricate network of electrical signals that are responsible for
coordinating muscles, the senses, speech, memories, thought and
emotion. Several diseases that directly affect the nervous system
have a genetic component: some are due to a mutation in a single
gene, others are proving to have a more complex mode of
inheritance. As our understanding of the pathogenesis of
neurodegenerative disorders deepens, common themes begin to emerge:
Alzheimer brain plaques and the inclusion bodies found in Parkinson
disease contain at least one common component, while Huntington
disease, fragile X syndrome and spinocerebellar atrophy are all
`dynamic mutation` diseases in which there is an expansion of a DNA
repeat sequence. Apoptosis is emerging as one of the molecular
mechanisms invoked in several neurodegenerative diseases, as are
other, specific, intracellular signaling events. The biosynthesis
of myelin and the regulation of cholesterol traffic also figure in
Charcot-Marie-Tooth and Neimann-Pick disease, respectively.
[0082] As used herein, "a signal disease or disorder" refers to a
pathological condition caused by defects in the signal transudation
process. Signal transudation within and between cells mean that
they can communicate important information and act upon it.
Hormones released from their site of synthesis carry a message to
their target site, as in the case of leptin, which is released from
adipose tissue (fat cells) and transported via the blood to the
brain. Here, the leptin signals that enough has been eaten. Leptin
binds to a receptor on the surface of hypothalamus cells,
triggering subsequent intracellular signaling networks.
Intracellular signaling defects account for several diseases,
including cancers, ataxia telangiectasia and Cockayne syndrome.
Faulty DNA repair mechanisms are also invoked in pathogenesis,
since control of cell division, DNA synthesis and DNA repair all
are inextricably linked. The end-result of many cell signals is to
alter the expression of genes (transcription) by acting on
DNA-binding proteins. Some diseases are the result of a lack of or
a mutation in these proteins, which stop them from binding DNA in
the normal way. Since signaling networks impinge on so many aspects
of normal function, it is not surprising that so many diseases have
at least some basis in a signaling defect.
[0083] As used herein, "a transporter disease or disorder" refers
to a pathological condition caused by defects in a transporter,
channel or pump. Transporters, channels or pumps that reside in
cell membranes are key to maintaining the right balance of ions in
cells, and are vital for transmitting signals from nerves to
tissues. The consequences of defects in ion channels and
transporters are diverse, depending on where they are located and
what their cargo is. For example, in the heart, defects in
potassium channels do not allow proper transmission of electrical
impulses, resulting in the arrhythmia seen in long QT syndrome. In
the lungs, failure of a sodium and chloride transporter found in
epithelial cells leads to the congestion of cystic fibrosis, while
one of the most common inherited forms of deafness, Pendred
syndrome, looks to be associated with a defect in a sulphate
transporter.
[0084] As used herein, high-throughput screening (HTS) refers to
processes that test a large number of samples, such as samples of
diverse chemical structures against disease targets to identify
"hits" (see, e.g., Broach, et al., High throughput screening for
drug discovery, Nature, 384:14-16 (1996); Janzen, et al., High
throughput screening as a discovery tool in the pharmaceutical
industry, Lab Robotics Automation: 8261-265 (1996); Fernandes, P.
B., Letter from the society president, J. Biomol. Screening, 2:1
(1997); Burbaum, et al, New technologies for high-throughput
screening, Curr. Opin. Chem. Biol., 1:72-78 (1997)]. HTS operations
are highly automated and computerized to handle sample preparation,
assay procedures and the subsequent processing of large volumes of
data.
[0085] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections that follow.
[0086] B. Methods for Detecting Protein Modification
[0087] In one aspect, the present invention is directed to a method
for detecting protein modification of a target protein in a sample,
which method comprises: a) contacting a sample containing or
suspected of containing a target protein with a capture molecule
immobilized on a solid support, said capture molecule is capable of
specifically binding to said target protein, whereby said target
protein is immobilized on said solid support; and b) assessing
modification status and/or identity of said immobilized target
protein.
[0088] Although the present method can be used to assess the
protein modification status of a single target protein at a time,
the present method is preferably used in a high-throughput format.
For example, the protein modification status (or profile) of a
plurality of target proteins can be assessed simultaneously by
contacting the plurality of target proteins with an immobilized
capture molecule, or a plurality of immobilized capture molecules,
simultaneously. Alternatively, the protein modification status (or
profile) of a single target protein can be assessed simultaneously
by contacting a target protein with a plurality of immobilized
capture molecules simultaneously. Preferably, the protein
modification status (or profile) of a plurality of target proteins
can be assessed simultaneously by contacting the plurality of
target proteins with.
[0089] The protein modification status (or profile) of any
plurality, i.e., group, of target proteins can be assessed by the
present method. Preferably, the protein modification status (or
profile) of a group of structurally and/or functionally related
proteins are assessed. The "group of structurally and/or
functionally related proteins" can be a group of proteins, at their
natural status, that are structurally linked, located at the same
cellular locations, e.g., cellular organelles, located in the same
tissues or organs, expressed and/or be functional in the same
biological stages, e.g., a particular cell cycle stage or
developmental stage, or expressed and/or be functional in the same
biological pathway, e.g., a particular metabolism pathway, signal
transduction pathway, or act as a regulator for a pathway
activation or a biological function etc.
[0090] In a specific embodiment, the group of structurally and/or
functionally related proteins is a group of proteins, at their
natural status, that are located in the same cellular organelles.
Non-limiting examples of such cellular organelles include nucleus,
mitochondria, chloroplasts, ribosomes, ERs, Golgi apparatuses,
lysosomes, proteasomes, secretory vesicles, vacuoles or microsomes,
cytoplasm and other plasms within the such cellular organelles.
[0091] In another specific embodiment, the group of structurally
and/or functionally related proteins is located in the same tissues
or organs. Exemplary tissues include connective, epithelium, muscle
or nerve tissues. Exemplary organs include an accessory organ of
the eye, annulospiral organ, auditory organ, Chievitz organ,
circumventricular organ, Corti organ, critical organ, enamel organ,
end organ, external female gential organ, external male genital
organ, floating organ, flower-spray organ of Ruffini, genital
organ, Golgi tendon organ, gustatory organ, organ of hearing,
internal female genital organ, internal male genital organ,
intromittent organ, Jacobson organ, neurohemal organ,
neurotendinous organ, olfactory organ, otolithic organ, ptotic
organ, organ of Rosenmuller, sense organ, organ of smell, spiral
organ, subcommissural organ, subfomical organ, supernumerary organ,
tactile organ, target organ, organ of taste, organ of touch,
urinary organ, vascular organ of lamina terminalis, vestibular
organ, vestibulocochlear organ, vestigial organ, organ of vision,
visual organ, vomeronasal organ, wandering organ, Weber organ and
organ of Zuckerkandl can be manipulated. Exemplary internal animal
organs include brain, lung, liver, spleen, bone marrow, thymus,
heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous
system, gland, internal blood vessels, etc can be manipulated.
[0092] In still another specific embodiment, the group of
structurally and/or functionally related proteins is located in the
same body fluid such as blood, urine, saliva, bone marrow, sperm or
other ascitic fluids, and subfractions thereof, e.g., serum or
plasma.
[0093] In yet another specific embodiment, the group of
structurally and/or functionally related proteins shares identical
or similar structural and/or functional characteristics, such as
nutrient or storage proteins, contractile or motile proteins,
structural proteins, defense proteins, or regulatory protein.
[0094] Any molecule, or complex or combination therefor, that is
capable of specifically binding to a target protein, or one or more
member(s) of a plurality of target proteins, can be used as the
capture molecule in the present method. In one specific embodiment,
the capture molecule is capable of specifically binding to both a
modified and an unmodified forms of a target protein. In another
specific embodiment, the capture molecule is capable of
specifically binding to a modified form of the target protein but
is not capable of specifically binding to an unmodified form of the
target protein. In a preferred embodiment, the capture molecule is
an antibody, e.g., a polyclonal antibody, a monoclonal antibody, an
antibody fragment retaining its desired binding specificity, or a
combination thereof.
[0095] The capture molecule can be macromolecules such as peptides,
proteins, e.g., antibodies or receptors, oligonucleotides, nucleic
acids, e.g., protein-binding DNA or RNA molecules, vitamins,
oligosaccharides, carbohydrates, lipids, or small molecules, or a
complex thereof.
[0096] Any proteins or peptides that are capable of specifically
binding to a target protein, or one or more member(s) of a
plurality of target proteins, can be used as the capture molecule
in the present method. For example, enzymes, transport proteins
such as ion channels and pumps, nutrient or storage proteins,
contractile or motile proteins such as actins and myosins,
structural proteins, defense protein or regulatory proteins such as
antibodies, hormones and growth factors can be used.
[0097] Any nucleic acids, including single-, double and
triple-stranded nucleic acids, that are capable of specifically
binding to a target protein, or one or more member(s) of a
plurality of target proteins, can be used as the capture molecule
in the present method. Examples of such nucleic acids include DNA,
such as A-, B- or Z-form DNA, and RNA such as mRNA, tRNA and
rRNA.
[0098] Any vitamins that are capable of specifically binding to a
target protein, or one or more member(s) of a plurality of target
proteins, can be used as the capture molecule in the present
method. For example, water-soluble vitamins such as thiamine,
riboflavin, nicotinic acid, pantothenic acid, pyridoxine, biotin,
folate, vitamin B,.sub.2 and ascorbic acid can be manipulated.
Similarly, fat-soluble vitamins such as vitamin A, vitamin D,
vitamin E, and vitamin K can be used.
[0099] Any lipids that are capable of specifically binding to a
target protein, or one or more member(s) of a plurality of target
proteins, can be used as the capture molecule in the present
method. Examples of lipids include triacylglycerols such as
tristearin, tripalmitin and triolein, waxes, phosphoglycerides such
as phosphatidylethanolamine, phosphatidylcholine,
phosphatidylserine, phosphatidylinositol and cardiolipin,
sphingolipids such as sphingomyelin, cerebrosides and gangliosides,
sterols such as cholesterol and stigmasterol and sterol fatty acid
esters. The fatty acids can be saturated fatty acids such as lauric
acid, myristic acid, palmitic acid, stearic acid, arachidic acid
and lignoceric acid, or can be unsaturated fatty acids such as
palmitoleic acid, oleic acid, linoleic acid, linolenic acid and
arachidonic acid.
[0100] Any suitable solid support can be used in the present
method. In one example, the solid support can be a silicon,
plastic, nylon, glass, ceramic, photoresist, rubber or polymer
support. The solid support can be in any kind of suitable geometric
forms, e.g., a flat support, a set of sticks, or a set of beads.
Exemplary flat supports can comprise a slide, a chip, a filter, or
a membrane. Solid supports and methods for immobilizing desired
capture molecules on the solid supports that are known in the art
can be used. Preferably, solid supports and methods for
immobilizing desired capture molecules on the solid supports that
are described in the following Section C can be used in the present
method.
[0101] Any protein modification, especially post-translational
protein modification, can be assessed by the present method.
Exemplary protein modifications that can be assessed by the present
method include phosphorylation, acetylation, methylation,
ADP-ribosylation, addition of a polypeptide side chain, addition of
a hydrophobic group, and addition of a carbohydrate. In one
specific embodiment, the phosphorylation to be assessed is
phosphorylation on tyrosine, serine, threonine or histidine
residue. In another specific embodiment, the addition of a
polypeptide side chain to be assessed is the addition of ubiquitin.
In still another specific embodiment, the addition of a hydrophobic
group to be assessed is the addition of a fatty acid, e.g.,
myristate or palmitate, addition of an isoprenoid, e.g., farnesyl
or genranylgenranyl, or addition of a glycosyl-phosphatidyl
inositol anchor, e.g., a carbohydrate group comprises glycosyl.
[0102] Phosphorylation
[0103] Phosphorylation can include phosphorylation of a tyrosine,
serine, threonine or histidine. Antibodies that can be used to
detect these modifications can include phosphotyrosine-specific
antibody, phosphoserine-specific antibody, phosphoserine-specific
antibody, and phospho-threonine-proline antibody, for example.
Antibodies that can be used to detect these modifications also
include an antibody specific to a phosphorylated residue of a
protein such as phosphorylated c-Jun at Ser 73. Among various
post-translational modifications, protein phosphorylation was found
to be the most common mechanism for switching a protein from its
active state to an inactive state. The protein phosphorylation
includes tyrosine phosphorylation, serine/threonine phosphorylation
and histidine phosphorylation. The phosphorylation of p44/42 MAP
Kinase (Thr202/Tyr204) and MEKI/2 (Ser217/221) has been found to
contribute to the activation of mitogenic signal pathway. The
phosphorylation of SAPK/JNK (Thr183/Tyr185), p38 MAP kinase
Thr180/Tyr182, MKK3/MKK6 (Ser189/207), SEK1/MKK4 (Thr223) has been
found to contribute to the activation of stress signal pathway. The
phosphorylation of Akt (Ser473), Bad (Ser112/136) and p70 S6 Kinase
(Ser411, Thr421/Ser424) has been found to promote cell survival and
prevent cell apoptosis.
[0104] The phosphorylation of pathway-specific transcription
factors also can serve as a reliable marker for pathway activation.
For example, phosphorylation of ikb-a indicates activation of NFkB
signal pathway ( ); phosphorylation of ELK1, CREB, Ets1, Ets2, CBP,
PEA.sub.3, p.sub.90rSk and CEBP indicates activation of
mitogenic/differentiation signal pathway ( ); phosphorylation of
c-Jun, Elk1, ATF2, c-myc, SAP1a and PEA3 indicates activation of
cytoskeletal organization signal pathway ( ); phosphorylation of
ATF1, ATF2, Elk1, Max, CHOP, CREB, SAP1a and MAPKAPK-2 indicates
activation of apoptosis/stress signal pathway. Several ErbB family
receptors are implicated in tumor formation and these receptors are
activated through self tyrosine phosphorylation.
[0105] A protein array for tyrosine phosphorylation can contain
antibodies that can specifically capture proteins whose tyrosine
phosphorylated form are of interest. After capturing those proteins
on the membrane, an antibody against phosphorylated tyrosine is
used to detect the amount of phosphorylated form of each protein. A
protein array for tyrosine phosphorylation can be designed for
analyzing a groups of proteins involved in mitogenic signal
pathway, and can contain antibodies against EGF receptor, PDGF
receptor, SOS, Src, p44/4.sup.2 MAP Kinase. Another protein array
for tyrosine phosphorylation can contain antibodies against all
four members of ErbB family receptors: EGFR, ErbB-2, ErbB-3 and
ErbB-4.
[0106] To monitor activation state of mitogenic signal pathway,
stress signal pathway and a pptosis/survival signal pathway, a
protein array for phosphorylation can be designed for analyzing
phosphorylation state of pathway-specific kinases. These kinases
can include p44/42 MAP Kinase, MEK1/2, SAPK/JNK, p38 MAP kinase,
MKK3/MKK6, SEK1/MKK4, Akt, Bad and p70 S6 Kinase. Alternatively, a
protein array for phosphorylation can be designed for analyzing
phosphorylation state of pathway-specific transcription factors as
listed above.
[0107] Acetylation
[0108] Acetylation can be detected by use of an acetylated-lysine
antibody. Acetylation of p53 is associated with a change of its
transcriptional activity after DNA damage. Acetylation of histone
H3 is increased in response to DNA damage and mitogen stimulation.
A protein array comprising acetylation can be used to
simultaneously analyze multiple proteins for their acetylation
status in a single assay. Such an array can include capture
molecules for p53 and various histones including histone 1, 2A, 2B,
3 and 4, and using acetylation detection antibodies the
modification on these capture molecules can be detected.
[0109] Methylation
[0110] Methylation specific antibodies can be used to detect
proteins having a methylation on one or more amino acids of a
polypeptide sequence of the protein. Detecting methylation may be
useful in a variety of biological contexts, for example, in human
neutrophils and other cell types, Ras-related guanosine
triphosphate-bin ding proteins are directed toward their regulatory
targets in membranes by a series of posttranslational modifications
that include methyl esterification of a carboxyl-terminal
prenylcysteine residue. The amount of carbosyl methylation of
Ras-related proteins increased in response to the chemoattractant
N-formyl-methionyl-leucyl-phenylalanine (FMLP). Activation of
Ras-related proteins by guanosine-5'-O-(3-thiotriphosphate) has a
similar effect and induced translocation of p22rac2 from cytosol to
plasma membrane. Inhibitors of prenylsysteine carbosyl methylation
effectively block neutrophil responses to FMLP. A protein array for
Ras-related proteins for their methylation can be used to
simultaneously analyze methylation status of multiple Ras-related
proteins in a single assay. These Ras-related proteins can include,
for example, H-Ras, K-Ras, R-Ras, RhoA, Rac1, Rac2, Ra1, etc.
Antibodies (capture molecules) against each of Ras-related proteins
are spotted each on its own specific area of a protein array. The
Ras-related proteins of a protein sample are captured and then a
detection antibody specific for methylation (an anti-methylation
antibody) contacts the captured molecules to detect those proteins
that have been methylated.
[0111] ADP-Ribosylation
[0112] ADP-ribosylation specific antibodies can be used to detect
proteins having an ADP ribosylation modification. Detecting ADP
ribosylation can be useful in a variety of biological contexts,
including, for example in the case of when cholera toxin induces
activation of adenylate cyclase from small intestinal epithelium.
The activation is associated with ADP-ribosylation of a number of
proteins including Gs alpha subunit and a 40kd, a 45 kd and a 47 kd
proteins located in the bruch-border membrane. A specialized
ADP-ribosylation protein array can be made for studying ADP
ribosylation of these cholera toxin related proteins. Antibodies
(capture molecules) against each of these proteins can be spotted
each on its own specific area of a protein array. The protein array
is used to capture these proteins from a sample of proteins and the
captured proteins are detected for their ADP ribosylation through
contact with an anti-ADP-ribosylation antibody (the detection
molecule). Another example of use of a protein array for ADP
ribosylation is in the case of poly-ADP ribosylation of histones
which is found to increase significantly in mitogen-activated
lymphoid cells. A specialized histone poly-ribosylation protein
array can be used to simultaneously analyze poly-ribosylation of
various histones. Such a protein array can contain antibodies
(capture molecules) against various histones such as, for example,
histone 1, histone 2A, histone 2B, histone 3 and histone 4. The
protein array is used to capture various histones from a sample of
proteins and the target proteins are detected for their ADP
ribosylation through contact with an anti-ADP-ribosylation antibody
or anti-poly-ADP ribosylation antibody.
[0113] Polypeptide Chain Addition
[0114] An example of addition of a polypeptide chain is
ubiquitination. Detection of ubiquitination on a target protein can
be made using an ubiquitin-specific antibody or
polyubiquitin-specific antibody for example. Ubiquitination
involves the covalent attachment of ubiquitin, an evolutionary
highly conserved 76-amino acid polypeptide which is abundantly
present in all eukaryotic cells to the 1-amino group of one or more
lysine side chains of target proteins. Ubiquitination-dependent
degradation is involved in the degradation of proteins that are
either damaged or no longer needed. Ubiquination plays important
role in cell regulation and signal transduction. Protein
ubiquitination can be a reversible process and is controlled by two
classes of enzymes: ubiquitin conjugating enzymes and
deubiquitinating enzymes. Recently, ubiquitination is found to be
responsible for the cell cycle-specific degradation of cyclins and
cytokine-induced breakdown of the transcription factor inhibitor
IkB. In addition, ubiquitination is responsible for the degradation
of several transcriptional factors including c-Jun, ATF2, Jun B and
p53. Phosphorylation of these transcriptional factors prolongs
their half-life significantly by blocking their ubiquitination. A
protein array for ubiquitination can be designed for analyzing a
group of transcriptional factors involved in stress signal pathway.
Such a protein array can contain antibodies against, e.g. c-Jun,
ATF-2, Jun B and p53 etc. A protein array for ubiquitination can
contain antibodies against cancer related proteins such as, e.g.
p53, b-catenin and cyclin D1 etc.
[0115] Hydrophobic Group Addition
[0116] Addition of a hydrophobic group to a protein is another type
of protein modification that affects the biological activity in a
cell. Several hundreds of cellular and viral proteins are now known
to be covalently modified by lipophilic moieties. These proteins
include proteins, guanine nucleotide binding proteins,
transmembrane receptors, and viral structural proteins. Among the
most common hydrophobic modifications are fatty acids (myristate
and palmitate), isoprenoids (farnesyl and geranylgeranyl), and
glycosyl-phosphatidyl inositol anchors. The lipid attachment to
these proteins influences protein-protein interactions, membrane
binding affinity, and cellular signal transduction by the modified
proteins. For example, a Src mutant devoid of myristoylation and a
Ras mutant devoid of prenylation abrogate their cellular
transformation ability. While most of lipophilic phosphorylation is
irreversible, palmitoylation is a reversible process. Bradykinin
treatment induces depalmitoylation of nitric oxide synthase and
translocation of the depalmitoylated protein from membrane to
cytosol. Lipophilic modification of proteins are often studied by
metabolic labeling cells with a radioactive lipid subunit.
Recently, lipid specific antibody such as anti-farnesyl antibody is
used to identify the presence of lipophilic modification. A protein
array for lipophilic modification can contain an array of
antibodies each capable of capturing a protein of interest from a
cell lysate for analyzing the presence of a specific lipid by a
second antibody specific against the lipid of interest.
[0117] Another example of addition of a hydrophobic group is
addition of an isoprenoid. For example, a protein array to detect
such geranylgeranylation can be designed for analyzing a group of
small guanine nucleotide binding proteins for the presence of
genranylgeranylation. A geranylgeranylation protein array can
contain antibodies (capture molecules) against Rap1A/Krev1, Rac,
Ral and Rho etc. A protein array for farnesylation can be designed
for analyzing a group of proteins for the presence of
farnesylation. A farnesylation protein array can contain antibodies
against H-Tas, N-Ras, K-Ras, Lamins A, Lamin B, transductin .tau.
subunit, and Rhodopsin kinase.
[0118] Carbohydrate Addition
[0119] Addition of a carbohydrate to a polypeptide sequence of a
protein can include, for example, glycosylation. Protein
glycosylation is an important post-translational modification
occurred in the lumen of the rough endoplasmic reticulum and in the
Golgi. To date, there are estimates of over 200 glycosyltransferase
enzymes involved in the addition of sugars onto newly synthesized
proteins. Specific carbohydrate structures participate in cell-cell
and cell-substratum interactions affecting processes such as
lymphocyte trafficking, immune cell stimulation, embryogenesis, and
cancer metastasis. Lectins as molecules can specifically recognize
a certain structure of carbohydrate group attached to proteins.
Antibodies against a specific carbohydrate group are also developed
to detect the presence of the modified group on the protein of
interest. For example, monoclonal antibody (Mab) 3E1.2 binds to
multimers of the sialylated carbohydrate in a protein
conformation-dependent manner on human mucins. The antigen for Mab
3E1.2 is elevated in breast cancer patients. Alteration of
carbohydrate groups on proteins are found-to be associated with
various cancers. Many of these alterations can be detected by
lectins or carbohydrate-specific antibody. A protein array for
glycosylation can contain an array of antibodies each capable of
capturing a protein of interest from a sample of proteins for
analyzing the presence of a specific carbohydrate group by a second
carbohydrate-specific antibody (e.g. a polysaccharide-specific
antibody) or a lectin specific for the particular carbohydrate
group.
[0120] The present method can be used in different formats. For
example, the target protein can be immobilized via the specific
interaction between a capture molecule and the peptidic portion of
the target protein, and then the protein modification status of the
immobilized target protein is assessed. Alternatively, the target
protein can be immobilized via the specific interaction between a
capture molecule and the modification moiety or the combination of
the modification moiety and the peptidic portion of the target
protein, and then the identity of the of the immobilized target
protein is assessed. Accordingly, in one specific embodiment, the
target protein is first immobilized by a capture molecule that is
capable of specifically binding to both a modified and an
unmodified forms of a target protein, and then the modification
status of the immobilized target protein is assessed by contacting
the immobilized target protein with a detection molecule that is
capable of specifically binding to the modified target protein but
is not capable of specifically binding to the unmodified target
protein itself. In this embodiment, the modification status of the
immobilized target protein can also be determined by other suitable
physical or chemical means (See e.g., Yan et al., J. Chromatogr.
A., 808(1-2):23-41 (1998). For example, the physical or chemical
means can comprise chemical or radioisotopic label of the protein
modification moiety. Alternatively, the physical or chemical means
can comprise any suitable analytical means, e.g., chromatographic,
electrophoretic, protein sequencing, mass spectrometry and NMR
means, for detecting the protein modification moiety. In another
specific embodiment, the target protein is first immobilized by a
capture molecule that is capable of specifically binding to a
modified form of the target protein but is not capable of
specifically binding to an unmodified form of the target protein,
and then the identity of the immobilized target protein is assessed
by contacting the immobilized target protein with a detection
molecule that is capable of specifically binding to the unmodified
target protein itself but is not capable of specifically binding to
the modified target protein.
[0121] In a specific embodiment, two or more capture molecules are
immobilized onto the solid support. A capture molecule can be, e.g.
an antibody specific for a target protein, but may also be a
non-antibody molecule, e.g. a lectin, or other protein,
polypeptide, or peptide specific for a target molecule. The capture
molecule may also be a non-protein molecule, for example a small
molecule, nucleic acid, polynucleotide or other type of molecule
capable of being immobilized onto the solid support and also
capable of binding a target protein with some affinity and
specificity. A solid support (e.g. a slide, wafer, membrane or
filter) will have a variety of spots or positions on which
populations of the same capture molecules can be placed. At each
spot or location many molecules of a particular capture molecule
can be immobilized. The amount of capture molecules required for
detecting an amount of bound target protein will depend on the
detection system being used (i.e. the more sensitive the detection
system, the less capture molecules needed and the less capture
molecule-target protein binding pairs will be generated and/or
needed for detection), on the binding affinity between the capture
molecule and the target protein, the expected relative amount of
target protein in the protein sample, and other considerations. The
amount of capture protein should be sufficient to generate
detectable signal by the conventional means used in the laboratory.
To date, the detection sensitivity for radioactive .sup.32P and
fluorescence dye such as DBCI (a dicarbocyanine analog of
indocyanine green) is 100,000 molecules. Therefore, the amount of
the capture protein should be greater than 100,000 if the
modification specific antibody is directly linked with a single
radioactive 32P molecule or DBCI molecule (Silzel J, et al, 1998,
Clinical Chemistry 44:2036-2043). However, less amount of the
capture protein can be used if the modification specific antibody
is linked with multiple detection molecules (32P, DBCI, etc.)
through direct conjugation or enzyme amplification (see below for
additional information). The size of the spot can be in the range
of about 5 um to about 1 cm in diameter, for example. The amount of
the capture protein "will depend on the size of the spot and the
linear range of detection assay. Various means can be used to spot
the capture molecules. For preparing a protein microarray (spot
size is <1 mm in diameter), mechanical microspotting and ink
jetting is preferred to be used (Shena M, et al, 1998, TIBTECH
16:302-306). For preparing protein array (spot size is >1 mm in
diameter), spotting can be achieved through using conventional lab
pipette.
[0122] The target molecule will preferably be bound to a capture
molecule at an epitope or site of the target molecule that leaves
any modification moiety on the target protein available for binding
a detection molecule later. A capture molecule is selected for the
specificity and affinity for binding particular target protein. The
capture molecule can be immobilized on the solid support by
following a procedure, for example, as follows: first blocking the
protein array with blocking reagents (e.g. dry milk, gelatin or BSA
containing solution) followed by rinsing away the blocking reagents
using e.g., TBST or PBST. The protein array is then incubated with
biologically activated sample such as cell lysate and tissue lysate
etc. for a few hours. Proteinase inhibitors and phosphate
inhibitors are usually included in the lysates. After the
incubation, the protein array is then washed with TBST or PBST
followed by incubation with modification-specific antibody for
around 1 hour or so. The protein array is further washed with TBST
or PBST and subjected to appropriate procedure for developing
detection signals.
[0123] Once the capture molecules are immobilized on the solid
support, the solid support is contacted with a biologically active
sample of proteins comprising target proteins that may have
undergone the subject protein modification. The target proteins
specific for a given population of capture molecules bind the
capture molecules and remain bound after washing. The detection can
be achieved through linking modification-specific antibody with
detection molecules such as fluorescent molecules or enzymes that
are capable of depositing substrates such as fluorescent molecules,
chromogenic substrates, and chemiluminecent substrates.
Modification-specific antibody can also be linked to detection
molecules indirectly through molecules such as biotin or other
haptens such as fluorescein and digoxigenin etc. for amplification.
Enzymes-linked strepavidin or enzyme linked-antibody against the
hapten is then used for the detection. To achieve a greater
amplification, the substrate for the enzymes can be linked to
molecules such as biotin or other haptens such as fluorescein and
digoxigenin etc., followed by detection by enzymes-linked
strepavidin or enzyme linked-antibody against the hapten.
[0124] The bound target proteins are then contacted with detection
molecules specific for the subject protein modification. All the
different target proteins will be screened for the same protein
modification. The detection molecule can be an antibody or other
binding molecule specific for the modification being detected. The
antibody can be a part of an antibody, for example, a polypeptide
having specificity for the modification on the target proteins that
are bound to capture molecules on the solid support. The detection
molecules may be conjugated themselves to a detection means (e.g.
enzymatic, fluorescent, chemiluminescent detection means), or may
themselves need to contact a label or tag that is detectable. The
tag, label, or detection means on the detection molecule can be,
for example, a color tag, an oligo tag, a fluorescent tag, or a
radio tag, etc. Where antibodies are used to detect the bound
proteins, for example, a detection-ready antibody can be, e.g.
conjugated with enzymes such as alkaline phosphatase (AP), horse
radish peroxidase (HRP) or others for direct detection or
conjugated with linker molecule such as biotin or other for
subsequent linking to detection molecules.
[0125] For example, a modification specific antibody can bind an
antigen captured on the solid support. From there a monoclonal
antibody linked with a detection molecule or molecules binds the
first antibody. Fluorescence molecules can then be detected without
amplification of the signal. Alternatively, enzymes can be added
that provide amplification of the signal, and then either
fluorescent molecules, chromogenic substrates, or chemiluminescent
substrates are detected. Another option for detection can be that a
second monoclonal antibody binds the first monoclonal antibody, but
this second monoclonal antibody is linked to indirect molecules
such as, e.g. biotin, or other haptens such as fluorescein, or
digoxigenin, etc. These indirect molecules react with a strepavidin
or hapten antibody linked to enzymes. Detection then proceeds from
either fluorescence molecules as a substrate, chromogenic molecules
as a substrate, or chemiluminescent molecules as a substrate for
the enzyme. Alternatively, for greater amplification, these enzymes
can react upon substrates linked to another amplification molecule,
e.g. biotin, fluorescein, or digioxigenin, etc., which in turn
react with streptavidin or hapten antibody linked to enzymes that
then react with fluorescent, chromogenic or chemiluminescent
substrates. See Ausabel et al., eds., in the Current Protocol of
Molecular Biology series of laboratory technique manuals. 1987-1997
Current-Protocols, 1994-1997 John Wiley and Sons, Inc.
[0126] In another specific embodiment, the organic phosphate can be
converted into inorganic phosphate to detect inorganic phosphate in
situ on the array (Kates, Techniques of Lipidology, 3.sup.rd Ed.
(North-Holland/Arnericam Elsevier, New York)). In still another
specific embodiment, the phosphorylation substrates are left
unlabeled on the array. The substrates are phosphorylated using
adenosine 5'-O-(3-thiotriphosphate) (ATP.gamma.-S) instead of ATP.
After the kinase assay step, the thiophosphorylated product is then
reacted with iodoacetyl derivative of a tag compound. The tag
compound is either labeled with fluorecence, color or
chemiluminecence that provide detection mean for the amount of
phosphorylation (Jeong and Nikiforov, BioTechnique, 27:1232-1238
(1999)).
[0127] The protein modification status of a target protein, or a
plurality of target proteins, in any sample can be assessed by the
present method. Preferably, the sample to be assessed is a
biological sample, such as a biological fluid or a biological
tissue. Examples of biological fluids include urine, blood, plasma,
serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears,
mucus, amniotic fluid or the like. Biological tissues are
aggregates of cells, usually of a particular kind together with
their intercellular substance that form one of the structural
materials of a human, animal, plant, bacterial, fungal or viral
structure, including connective, epithelium, muscle and nerve
tissues. Examples of biological tissues also include organs,
tumors, lymph nodes, arteries and individual cell(s).
[0128] The protein modification status of any target protein, or
any plurality of target proteins, can be assessed by the present
method. Preferably, the target protein to be assessed is involved
in a biological pathway, belongs to a group of proteins with
identical or similar biological function, expressed in a stage of
cell cycle, expressed in a cell type, expressed in a tissue type,
expressed in an organ type, expressed in a developmental stage, a
protein whose expression and/or activity is altered in a disease or
disorder type or stage, or a protein whose expression and/or
activity is altered by drug or other treatments.
[0129] In a specific embodiment, the present method can be used to
assess protein modification profile of a particular tissue such as
epithelium tissue, connective tissues, including blood, bone, and
cartilage, muscle tissue and nerve tissue.
[0130] In another specific embodiment, the present method can be
used to assess protein modification profile of a particular organ,
i.e., any part of the body exercising a specific function, as of
respiration, secretion or digestion.
[0131] In still another specific embodiment, the present method can
be used to assess protein modification profile of a particular
organism, such as plant, animal, bacteria, e.g., eubacteria and
archaebacteria, virus, e.g., Classes I-VI viruses, or fungus.
[0132] In yet another specific embodiment, the present method can
be used to assess protein modification profile of a particular
disease or disorder", such as infection, neoplasm (neoplasia),
cancer, an immune system disease or disorder, a metabolism disease
or disorder, a muscle and bone disease or disorder, a nervous
system disease or disorder, a signal disease or disorder, or a
transporter disease or disorder.
[0133] In yet another specific embodiment, the protein samples can
come from tissues or cell lines, for example. The protein sample
can be from, e.g. an animal, plant, fungus (e.g. yeast) or
bacteria. The animal can be, e.g. a fish, amphibian, reptile,
insect (e.g. drosophila), or mammal. The mammal can be e.g. a
human, primate, dog, cat, rodent, goat, sheep, or cow.
[0134] Where a comparison is made using a protein array, e.g. a
comparison between a control array and an array from a protein
mixture of a particular condition or change in a condition, the
control sample can be e.g. from a normal tissue and the
experimental sample can be from changed tissue, e.g. diseased
tissue, or the control and test sample can be from the same tissue
or cells or from different animals; from the same tissues of a
different developmental stage. The organisms in a comparison can be
e.g. wild type, diseased, knockout or transgenic. The array could
also represent different tissues or cells from the same body. The
control sample can be, e.g. untreated cells and the experimental
sample can be treated cells. The treatment can be various
biological, physical or chemical treatments such as, for example,
any drug treatment whether a known approved drug or a test drug.
The treatment can also comprise such treatments, e.g. as
administration of a growth factor treatment, UV irradiation, or
other drugs, chemicals or therapies to treat a disease or condition
or to cause a change in a condition.
[0135] The biologically active sample of proteins can be, for
example contained in cell lysate. The proteins may be isolated from
multiple cells, populations of different cells, tissue, serum,
blood, body fluids, or other sources which potentially contain
modulated proteins and which it is desirable to test for the
protein modification that has occurred in the sample. Cell lysates
may be prepared as described in Ausabel et al., eds., in the
Current Protocol of Molecular Biology series of laboratory
technique manuals. 1987-1997 Current Protocols, 1994-1997 John
Wiley and Sons, Inc.
[0136] In another aspect, the present invention is directed to a
method for identifying biologically distinguishable marker(s)
associated with a biosample, which comprises: 1) assessing protein
modification profile of a biosample through the above-described
method; 2) assessing protein modification profile of a comparable
control biosample through the above-described method; and 3)
comparing the protein modification profile obtained in step 1) with
the protein modification profile obtained in step 2) to identify
biologically distinguishable protein modification profile marker(s)
associated with said biosample. Preferably, the identified
biologically distinguishable protein modification profile marker(s)
are indicative of the protein modification profile of the
biological source from which the biosample is derived.
[0137] C. Kits and Arrays for Detecting Protein Modification
[0138] In still another aspect, the present invention is directed
to a kit for detecting protein modification, which kit comprises:
a) a capture molecule immobilized on a solid support, said capture
molecule is capable of specifically binding to a target protein;
and b) means for assessing modification status and/or identity of
said target protein.
[0139] Although the present kit can be used to assess the protein
modification status of a single target protein at a time, the
present kit is preferably used in a high-throughput format. For
example, the kit can comprise a plurality of capture molecules that
is immobilized on the solid support, each of said capture molecules
is capable of specifically binding to a member protein of a group
of structurally and/or functionally related target proteins.
[0140] The protein modification status (or profile) of any
plurality, i.e., group, of target proteins can be assessed by the
present kit. Preferably, the protein modification status (or
profile) of a group of structurally and/or functionally related
proteins is assessed.
[0141] Any molecule, or complex or combination therefor, that is
capable of specifically binding to a target protein, or to one or
more member(s) of a plurality of target proteins, can be used as
the capture molecule in the present kit. In one specific
embodiment, the capture molecule is capable of specifically binding
to both a modified and an unmodified forms of a target protein. In
another specific embodiment, the capture molecule is capable of
specifically binding to a modified form of the target protein but
is not capable of specifically binding to an unmodified form of the
target protein. In a preferred embodiment, the capture molecule is
an antibody, e.g., a polyclonal antibody, a monoclonal antibody, an
antibody fragment retaining its desired binding specificity, or a
combination thereof.
[0142] Any suitable solid support can be used in the present kit.
In one example, the solid support can be a silicon, plastic, nylon,
glass, ceramic, photoresist, rubber or polymer support. The solid
support can be in any kind of suitable geometric forms, e.g., a
flat support, a set of sticks, or a set of beads. Exemplary flat
supports can comprise a slide, a chip, a filter, or a membrane.
[0143] Any protein modification, especially post-translational
protein modification, can be assessed by the present kit. Exemplary
protein modifications that can assessed by the present method
include phosphorylation, acetylation, methylation,
ADP-ribosylation, addition of a polypeptide side chain, addition of
a hydrophobic group, and addition of a carbohydrate. In one
specific embodiment, the phosphorylation to be assessed is
phosphorylation on tyrosine, serine, threonine or histidine
residue. In another specific embodiment, the addition of a
polypeptide side chain to be assessed is the addition of ubiquitin.
In still another specific embodiment, the addition of a hydrophobic
group to be assessed is the addition of a fatty acid, e.g.,
myristate or palmitate, addition of an isoprenoid, e.g., farnesyl
or genranylgenranyl, or addition of a glycosyl-phosphatidyl
inositol anchor, e.g., a carbohydrate group comprises glycosyl.
[0144] The present kit can be used in different formats. For
example, the target protein can be immobilized via the specific
interaction between a capture molecule and the peptidic portion of
the target protein, and then the protein modification status of the
immobilized target protein is assessed. Alternatively, the target
protein can be immobilized via the specific interaction between a
capture molecule and the modification moiety or the combination of
the modification moiety and the peptidic portion of the target
protein, and then the identity of the of the immobilized target
protein is assessed. Accordingly, in one specific embodiment, the
target protein is first immobilized by a capture molecule that is
capable of specifically binding to both a modified and an
unmodified forms of a target protein, and then the modification
status of the immobilized target protein is assessed by contacting
the immobilized target protein with a detection molecule that is
capable of specifically binding to the modified target protein but
is not capable of specifically binding to the unmodified target
protein itself. In this embodiment, the modification status of the
immobilized target protein can also be determined by other suitable
physical or chemical means. For example, the physical or chemical
means can comprise chemical or radioisotopic label of the protein
modification moiety. Alternatively, the physical or chemical means
can comprise any suitable analytical means, e.g., chromatographic,
electrophoretic, protein sequencing, mass spectrometry and NMR
means, for detecting the protein modification moiety. In another
specific embodiment, the target protein is first immobilized by a
capture molecule that is capable of specifically binding to a
modified form of the target protein but is nor capable of
specifically binding to an unmodified form of the target protein,
and then the identity of the immobilized target protein is assessed
by contacting the immobilized target protein with a detection
molecule that is capable of specifically binding to the unmodified
target protein itself but is not capable of specifically binding to
the modified target protein.
[0145] The protein modification status of a target protein, or a
plurality of target proteins, in any sample can be assessed by the
present kit. Preferably, the sample to be assessed is a biological
sample.
[0146] The protein modification status of any target protein, or
any plurality of target proteins, can be assessed by the present
kit. Preferably, the target protein to be assessed is involved in a
biological pathway, belongs to a group of proteins with identical
or similar biological function, expressed in a stage of cell cycle,
expressed in a cell type, expressed in a tissue type, expressed in
an organ type, expressed in a developmental stage, a protein whose
expression and/or activity is altered in a disease or disorder type
or stage, or a protein whose expression and/or activity is altered
by drug or other treatments.
[0147] In one specific embodiment, the kit can further comprise: a)
instructions for using the kit; b) reagents and buffers; and/or c)
a container(s) for the kit contents.
[0148] In yet another aspect, the present invention is directed to
an array of protein capture molecules, which array comprises: a) a
solid support; and b) a plurality of capture molecules immobilized
on said solid support, wherein each of said molecules is capable of
specifically binding to both a modified and an unmodified form of a
member protein of a plurality of target proteins. Preferably, the
plurality of target proteins comprises a group of structurally
and/or functionally related proteins.
[0149] The modified and unmodified forms of the same target protein
to be assessed by the present array can have same, but preferably,
different biological activities. The modified and unmodified forms
of the same target protein to be assessed by the present array can
represent same, but preferably, different physiological conditions
or biological statuses. The present array can be used to identify
pathway activation. The present array can also be used to identify
activation of a group of structurally and/or functionally related
protein. The present array can further be used to generate a
modification profile correlated to a physiological condition, drug
treatment and disease. The present array can also be used to
identify a physiological or pathological status. The present array
can also be used to record biological perturbation caused by drug
and other treatment.
[0150] In yet another aspect, the present invention is directed to
an array of protein capture molecules, which array comprises: a) a
solid support; and b) a plurality of capture molecules immobilized
on said solid support, wherein each of said molecules is capable of
binding to a specific epitope generated from modification of a
modified protein, e.g., Rb.
[0151] In a specific embodiment, the solid supports can be a
two-dimensional relatively flat surface. For example, the solid
support can be a slide, a wafer, a filter, or a membrane. These
supports can be made of any material to which the subject capture
molecules can be immobilized, and upon which the protein
modification may be detected with a detection molecule. Thus, for
example, the solid supports can be made of glass, plastic, polymer,
nylon, nitrocellulose, metal, or a favorable or useful mixture of
any of these materials. For a wafer or rectangular two dimensional
solid support, two or more populations of capture molecules are
immobilized onto the solid support at different distinct locations
in order to capture the target proteins and apply detection
molecules to screen for protein modification in those captured
target proteins.
[0152] The solid support may also be other than a rectangular two
dimensional surface, and may be in the form of multiple sticks
(e.g. flat sticks or strips, one each for a capture molecule
population). For a particular protein modification, where two or
more capture molecules are used, two or more sticks are made to
capture and detect the particular protein modification sought. The
solid support may also be collection of beads. A bead or group of
beads will be coated with a particular capture molecule, and the
entire population of beads will include beads specific for two or
more target proteins having the same-type protein modification.
Each bead will be coded or marked to be clear what target protein
is captured on the beads, and thus to detect which protein is
modulated. Both the bead and sticks may be made of any material
suitable for the purpose described including, e.g. glass, plastic,
polymer, nylon, nitrocellulose, metal, or a favorable or useful
mixture of any of these materials. The beads, sticks, strips, or
contiguous solid supports can be coded, e.g. with a bar code or ink
mark to identify the support, and to perhaps designate an
orientation, e.g. as with a contiguous solid support having a grid
of capture molecules. In any event, the practitioner needs to keep
track of what capture molecule is where on the contiguous solid
support, stick, strip, or bead, so that the target protein is
identifiable.
[0153] Two or more capture molecules are immobilized onto the solid
support. A capture molecule can be, e.g. an antibody specific for a
target protein, but may also be a non-antibody molecule, e.g. a
lectin, or other protein, polypeptide, or peptide specific for a
target molecule. The capture molecule may also be a non-protein
molecule, for example a small molecule, nucleic acid,
polynucleotide or other type of molecule capable of being
immobilized onto the solid support and also capable of binding a
target protein with some affinity and specificity. A solid support
(e.g. a slide, wafer, membrane or filter) will have a variety of
spots or positions on which populations of the same capture
molecules can be placed. At each spot or location many molecules of
a particular capture molecule can be immobilized. The amount of
capture molecules required for detecting an amount of bound target
protein will depend on the detection system being used (i.e. the
more sensitive the detection system, the less capture molecules
needed and the less capture molecule-target protein binding pairs
will be generated and/or needed for detection), on the binding
affinity between the capture molecule and the target protein, the
expected relative amount of target protein in the protein sample,
and other considerations. The amount of capture protein should be
sufficient to generate detectable signal by the conventional means
used in the laboratory. To date, the detection sensitivity for
radioactive 32P and fluorescence dye such as DBCI (a dicarbocyanine
analog of indocyanine green) is 100,000 molecules. Therefore, the
amount of the capture protein should be greater than 100,000 if the
modification specific antibody is directly linked with a single
radioactive 32P molecule or DBCI molecule (Silzel J, et al, 1998,
Clinical Chemistry 44:2036-2043). However, less amount of the
capture protein can be used if the modification specific antibody
is linked with multiple detection molecules (32P, DBCI, etc.)
through direct conjugation or enzyme amplification (see below for
additional information). The size of the spot can be in the range
of about 5 um to about 1 cm in diameter, for example. The amount of
the capture protein will depend on the size of the spot and the
linear range of detection assay. Various means can be used to spot
the capture molecules. For preparing a protein microarray (spot
size is <1 mm in diameter), mechanical microspotting and ink
jetting is preferred to be used (Shena M, et al, 1998, TIBTECH
16:302-306). For preparing protein array (spot size is >1 mm in
diameter), spotting can be achieved through using conventional lab
pipette.
[0154] The target molecule will preferably be bound to a capture
molecule at an epitope or site of the target molecule that leaves
any modification moiety on the target protein available for binding
a detection molecule later. A capture molecule is selected for the
specificity and affinity for binding particular target protein. The
capture molecule can be immobilized on the solid support by
following a procedure, for example, as follows: first blocking the
protein array with blocking reagents (e.g. dry milk, gelatin or BSA
containing solution) followed by rinsing away the blocking reagents
using e.g., TBST or PBST. The protein array is then incubated with
biologically activated sample such as cell lysate and tissue lysate
etc. for a few hours. Proteinase inhibitors and phosphate
inhibitors are usually included in the lysates. After the
incubation, the protein array is then washed with TBST or PBST
followed by incubation with modification-specific antibody for
around 1 hour or so. The protein array is further washed with TBST
or PBST and subjected to appropriate procedure for developing
detection signals.
[0155] In another specific embodiment, an array of immobilized
antibodies are used in the kits for detecting protein modification.
Any antibodies, whether polyclonal, monoclonal, single chain, Fc
fragment, Fab fragment, F(ab).sub.2 fragment, or a mixture thereof,
can be used to produce the antibody arrays.
[0156] The antibody array used in the kit can be produced on any
suitable solid surface, including silicon, plastic, glass, polymer,
such as cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene, ceramic, photoresist or rubber surface.
Preferably, the silicon surface is a silicon dioxide or a silicon
nitride surface. Also preferably, the array is made in a chip
format. The solid surfaces may be in the form of tubes, beads,
discs, silicon chips, microplates, polyvinylidene difluoride (PVDF)
membrane, nitrocellulose membrane, nylon membrane, other purous
membrane, non-porous membrane, e.g., plastic, polymer, perspex,
silicon, amongst others, a plurality of polymeric pins, or a
plurality of microtitre wells, or any other surface suitable for
immobilizing proteins and/or conducting an immunoassay.
[0157] The antibodies can be attached to the solid surface by any
methods known in the art (see generally, WO 99/39210, WO 99/40434).
For example, the antibodies can be attached directly or through
linker(s) to the surface. The antibodies can be attached to the
surface through non-specific, specific, covalent, non-covalent,
cleavable or non-cleavable linkage(s). The cleavable linkage can be
cleavable upon physical, chemical or enzymatic treatment. The
arrays can be arranged in any desired shapes such as linear,
circular, etc.
[0158] In one example, antibody array can be printed on a solid
surface using pins (passive pins, quill pins, and the like) or
spotting with individual drops of solution (WO 99/40434). Passive
pins draw up enough sample to dispense a single spot. Quill pins
draw up enough liquid to dispense multiple spots. Bubble printers
use a loop to capture a small volume which is dispensed by pushing
a rod through the loop. Microdispensing uses a syringe mechanism to
deliver multiple spots of a fixed volume. In addition, solid
supports, can be arrayed using piezoelectric (ink jet) technology,
which actively transfers samples to a solid support. In addition,
the methods disclosed in WO 95/35505 can also be used. The method
and apparatus described in WO 95/35505 can create an array of up to
six hundred spots per square centimeter on a glass slide using a
volume of 0.01 to 100 nl per spot. Suitable concentrations of
antibody range from about 1 ng/.mu.l to about 1 .mu.g/l. Further,
other methods of creating arrays, including photolithographic
printing (Pease, et a., PNAS 91(11):5022-5026, 1994) and in situ
synthesis can be used.
[0159] Methods for covalent attachment of antibodies to a solid
support are known in the art. Examples of such methods are found in
Bhatia, et al., Anal. Biochem. 178(2):408-413, 1989; Ahluwalia, et
al., Biosens. Bioelectron. 7(3):207-214, 1992; Jonsson, et al.,
Biochem. J. 227(2):373-378, 1985; and Freij-Larsson, et al.,
Biomaterials 17(22):2199-2207, 1996, all of which are incorporated
by reference herein in their entirety
[0160] Methods of reducing non-specific binding to a solid surface
are well known in the art and include washing the arrayed solid
surface with bovine serum albumin (BSA), reconstituted non-fat
milk, salmon sperm DNA, porcine heparin, and the like (see Ausubel,
et al., Short Protocols in Molecular Biology, 3rd ed. 1995).
[0161] D. Array, Kits and Methods for Detecting Enzymatic
Activity
[0162] In yet another aspect, the present invention is directed to
an array of enzyme substrates, which array comprises: a) a solid
support; and b) a plurality of substrates immobilized on said solid
support, wherein each of said substrates is a substrate of a member
enzyme of a group of structurally and/or functionally related
enzymes. Preferably, at least one of the member enzymes catalyzes a
protein modification reaction.
[0163] In yet another aspect, the present invention is directed to
a kit for detecting enzymatic activity, which kit comprises: a) an
array comprising a solid support, and a plurality of substrates
immobilized on said solid support, wherein each of said substrates
is a substrate of a member enzyme of a group of structurally and/or
functionally related enzymes; and b) means for assessing activity
of each of the member enzymes.
[0164] In yet another aspect, the present invention is directed to
a method for detecting enzymatic activity in a sample, which method
comprises: a) contacting a sample containing or suspected of
containing a group of structurally and/or functionally related
target enzymes with a plurality of substrates immobilized on a
solid support, wherein each of said substrates is a substrate of a
member enzyme of said group of target enzymes under conditions
suitable for said target enzymes to catalyze enzymatic reactions
involving said immobilized substrates; and b) assessing enzymatic
activities of said target enzymes. Preferably, at least one of the
target enzymes catalyzes a protein modification reaction.
[0165] The invention also provides a method of detecting enzymatic
activity in a biologically active or activated (e.g. also an
enzymatically active or activated) sample of proteins. The method
is practiced by providing a solid support comprising two or more
enzyme substrate molecules immobilized on the support each of which
can act as a substrate for an enzyme capable of the same enzymatic
modulation on a target substrate; contacting the solid support with
a biologically active sample comprising enzymatically active
proteins that may act on the immobilized substrates under enzymatic
conditions and perform a detectable enzymatic reaction on the
immobilized substrate; and detecting an enzyme modulation on the
substrate with a detection mean (such as 32P for phosphorylation)
or molecule that specifically binds the subject enzymatic
modulation in order to detect whether or not the sample of proteins
comprise a certain enzymatic activity, wherein the presence of a
particular enzymatic activity in a sample imparts information about
biological activity present in the sample. The enzymatic activity
can comprise an enzymatic activity selected from the group
consisting of kinase, phosphatase, transferase, lipid kinase,
isomerase, glycosidase, lipase, ligase, nuclease, peptidase,
protease, ubiquinase, glycosyltransferase and glycosylase.
[0166] The invention also includes a kit for performing the
detection of enzymatic activity in a biologically active or
activated sample of proteins. The kit comprises a solid support of
two or more enzyme substrate molecules immobilized on the support,
and reagents for practicing the method just described. The
invention also provides a solid support comprising two or more
enzyme substrate molecules immobilized on the support, each of
which can act as a substrate for an enzyme capable of the same
enzymatic modulation on a target substrate; wherein the solid
support provides an environment for an enzymatic reaction at the
immobilized substrates under enzymatic conditions, and further
wherein any modulation of the substrate by the enzyme can be
detected with a detection molecule under detection conditions on
the solid support.
[0167] E. Exemplary Methods, Kits Arrays and Uses Thereof
[0168] The following describes certain exemplary or preferred
methods, kits, arrays and their uses thereof.
[0169] To prepare a protein array on a two-dimensional surface,
capturing molecules at appropriate concentration are laid on a
specific location of the surface for a few hours or overnight for
absorption to the surface. The surface for absorption should
possess high protein-binding capability if the capturing molecule
is a protein. These surfaces include coated slides, nitrocellulose
or nylon membranes are commercially available: nitrocellulose or
nylon membrane and membrane slides can be from Schleicher &
Schuell (Keene, N.H.); glass slides can be purchased from Xenopore
(Hawthorne, N.J.).
[0170] For nitrocellulose and nylon membrane, for example,
capturing molecules can be coated by forced absorption onto the
membranes through vacuum. Bovine serum albumin (BSA) or milk
solution is then used to saturate the nonspecific binding of the
membrane to derive a prepared protein array. For glass slides the
antibody to be bound is simply dissolved in a buffer solution, e.g.
as described in the instructions from Xenopore (Hawthorne, N.J.).
At pH above the isoelectric point of the protein (important because
the binding takes place through the amine group on the protein, and
these must be in the free form for binding in the well) is
incubated for 2-3 hours at 37 degrees C., using 50 mM Na.sub.2
CO.sub.3/NaHCO.sub.3 solution of pH 9.6. This results in the
formation of a covalent bound between the surface and the protein.
The antibody solution is then incubated. Antibody cal also be
coated onto strepavidin coated slide through
biotin-conjugation.
[0171] To practice the invention, cell lysate can be prepared from
the source that needs to be studied. The source can be a cell line,
tissue or animal. Various methods have been used extensively for
the preparation of cell lysate. The cell lysate is then used to
incubate with a protein array. The protein array is a
two-dimensional surface or a population of beads or a population of
sticks. Each protein array could contain an array of antibodies
each with multiple copies spotted on a specific area of the
two-dimensional surface or a single type of bead or stick. After
the incubation of cell lysate with a protein array followed by a
few washing steps, proteins recognizing the antibodies on the
protein array are bound on the array. The bound protein array is
then stained with modification group specific antibodies (second
antibodies). The second antibody is preferred to be pre-linked with
a detection molecule such as a biotin, an enzyme, a dye or a
radioactive tag that becomes visible recognized by naked eyes after
development. The amount of the protein, modification of the protein
and conformation change of the protein is determined through the
detection system built on the second antibody. For the protein
array made on a two-dimensional surface, the location of capturing
antibody immobilized on the array serves as a reference for the
identity of detected proteins. For the protein array made on a
population of beads or sticks, a tag on each kind of bead or stick
serves as a reference for the identity of detected proteins. The
tag can be color, fluorescence, oligo, radiofrequency tag and other
tag that can be easily used to separate beads with different tags.
Protein array could also contain a population of enzyme substrates
that are used to determine activity of multiple enzymes
simultaneously, as described below.
[0172] The prepared protein array is incubated with a cell lysate
where proteins of interest are present for a period of time. The
incubation allows capture of proteins of interest onto the protein
array. The protein array is then washed several times by
appropriate solution (a washing solution can comprise TBS (50 mM
Tris, 125 mM NaCl, pH 7.4) and PBS (50 mM NaPO.sub.3, 125 mM NaCl)
with addition of detergent such as Tween-20 to become TBST and
PBST) a few times followed by incubating with a second antibody for
a period of time. After second antibody incubation, the protein
array is then washed a few times again with TBST to get rid of
nonspecific binding and develop chemiluminescence signal or
colormetric signal according to the detection system conjugated to
the second antibodies. If the second antibody is directly
conjugated by horseradish peroxidase (HRP) or alkaline phosphatase
(AP) enzymes, colormetric substrates can be used to develop signal.
If the second antibody is conjugated by biotin, for example, a
chemiluminescence detection system (Vector Laboratories,
Burlingame, Calif.) can be applied as well.
[0173] The methods, kits, and compositions of the invention can be
used, e.g. to monitor modification of group of proteins of
interest, by identifying affected proteins in, for example,
knockouts, transgenic animals, or microbile-infected or diseased
model animals. The invention can also be used to determine
molecular mechanisms of pathology in disease or tumor biology, to
discover the effect of therapeutic drugs or environmental toxins on
critical pathways, to understand the roles of critical genes in,
for example, cardiovascular and neurological disease, cancer,
toxicology, cell-cycle regulation, apoptosis, and stress response,
as a molecular diagnostic for identifying defective proteins, and
to classify disease type and disease stage in clinical samples.
[0174] Monitoring protein modification using protein arrays can be
accomplished in the context of, for example, a group of receptor
tyrosine kinases (RTK). Some tumor formations are associated with
activation of RTKs, such as EGFR family receptors). Tyrosine kinase
receptors can play a pivotal role in the formation, growth and
metastasis of human cancers. To date, more than thirty tyrosine
kinase receptors have been found belonging to six families
including EGF family, FGF family, PDGF family, insulin family, NGF
family and HGF family. Among these receptors, EGF family receptors,
FGF family receptors, PDGF receptors in PDGF receptor family, IGF
receptors in insulin receptor family and HGF receptor/Met in HGF
receptor family have been found to be involved in the tumor
formation. VEGF receptors in PDGF receptor family and FGF receptors
are involved in angiogenesis to support the growth of tumor and the
formation of metastasis. The signaling of HGF receptor/Met from HGF
receptor family also induce the invasiveness and metastatic
potential of various cell types. An RTK protein array provides an
efficient way for screening abnormally activated RTK in tumor
tissues. For RTK protein array, antibodies against RTKs are spotted
onto a surface and their activation is detected by
anti-phosphotyrosine antibody. Examples of arrays along these lines
can include, e.g. EGFR family protein array (EGFR, ErbB-2, ErbB-3
and ErbB-4); angiogenic RTK protein array (FGFR1, FGFR2, FGFR3,
FGFR4, FLT1, Flk1/KDR, FLT4); mitogenic RTK (EGFR, ErbB-2, ErbB-3
and ErbB-4, IGFR, PDGFR, FGFR1, FGFR2, FGFR3, FGFR4, FLT1,
Flk2/Flt3, Flk1/KDR, FLT4).
[0175] More specifically, a protein array for EGF family receptors
(also called the ErbB family of receptors) can be organized along
the following known biology of that receptor family. There are four
members: EGFR, ErbB-2, ErbB-3 and ErbB4. EGFR and ErbB-2/neu are
prototypes for a family of structurally related transmembrane
proteins that play a role in the development and progression of
cancer. Like other tyrosine kinase receptors, ErbB family receptors
are activated through tyrosine phosphorylation upon dimerization
induced by their ligand binding. The solid support can be contacted
with a cell lysates from, for example, breast cancer cell lines to
determine the identity and levels of activated EFG receptors in the
family that are present in any particular lysate.
[0176] A group of proteins involved in a signal transduction
pathway can be monitored using a protein array. For example see
Table I for p44/42 MAP kinase, p38 MAP kinase, JNK and Akt signal
pathways). The phosphorylation of proteins are detected by, e.g.
phosphotyrosine-specifi- c antibody, phosphoserine-specific
antibody, phosphoserine-specific antibody, and
phospho-threonine-proline antibody, or also for example, an
antibody specific to a phosphorylated residue of a protein such as
phosphorylated c-Jun at Ser 73.
[0177] A group of proteins involved in a common biological function
may also be monitored using a protein array. For example, the Stat
family of proteins can be monitored. Stat (signal transducers and
activators of transcription) are a class of transcription factors
that transmit signals for various cytokine and growth factors from
cytoplasm to nucleus. Stats are activated through phosphorylation.
There are 7 Stat family members to date. Stat 1 transmits signal
for IFN .alpha./.beta., IFN .alpha. and others; Stat 2 for IFN
.alpha./.beta.; Stat 3 for gp130 users and others; Stat 4 for
IL-12, IFN .alpha./.beta.; Stat 5a and Stat 5b for PRL, GH, EPO and
.gamma.c users; Stat 6 for IL-4 and IL13. In addition, some Stats
are also activated by other mitogenic signal and stress signal. For
example, Stat 3 is activated through p44/42 MAP kinase signaling
pathway and JNK signaling pathway. Stat1 can be also activated
through JNK signaling pathway and was found to play an important
role in inducing and maintaining constitutive levels of Caspases
for apoptosis. A Stat family protein array can contain antibodies
against each Stat and the activation of Stat can be detected by
phosphorylation specific antibodies, such as those described
herein.
[0178] A group of proteins whose modification is associated with a
certain condition of a type of cell or tissue can be monitored
using a protein array. The condition can be, for example, ischemia.
Ischemia is a medical condition that induces many cellular response
in brain. Many key regulators of various signaling pathways have
been activated by phosphorylation. They can include CREB, ATF2,
c-Jun, Jun B, Jun D, c-Fos, Rb, p44/42 MAP kinase, p38 MAP kinase,
JNKs. Ischemia condition protein array can include proteins whose
modification such as phosphorylation is associated with Ischemia
condition. This protein array can be used for classifying the
degree and type of Ischemia condition through generating a
fingerprint of phosphorylation status of those Ischemia related
proteins.
[0179] An array can be used to detect and/or monitor a group of
marker proteins whose modification is associated with a certain
condition such as a disease condition. For example a protein array
for typing human lung cancer can be constructed. Elevated tyrosine
phosphorylation of EGF receptor (EGF-r)(p 185), Erb B-2,
beta-catenin and p125FAK have been found to be associated with
human lung cancer. The elevation of tyrosine phosphorylation of
p125FAK is restricted to cancerous lung tissues and is closely
correlated with the nodal involvement of cancer and disease-free
survival time. A protein array for typing human lung cancer can
include antibodies against these four proteins and their tyrosine
phosphorylation status is determined by using anti-phosohotyrosine
antibody. This protein array can be used to classify different
stage of lung cancers into various category.
[0180] Other uses of the protein array methods, kits and
compositions can include identification of signal pathways.
Pathways affected in, for example, knockouts, transgenics,
microbile infected animals, or disease model animals can be
studied. Thus, an array can be used to determine molecular
mechanisms of pathology in disease and/or tumor biology, for
example; to discover the effect of therapeutic drugs or
environmental toxins on critical pathways; to understand the roles
of critical pathways in cardiovascular and neurological disease,
cancer, toxicology, cell-cycle regulation, apoptosis, and stress
response. It can also serve as molecular diagnostic mean for
identifying defected pathways.
[0181] A family of proteins possessing a similar biochemistry
property but involving different biological pathways can be studied
and/or monitored using a protein array. For example, an MKK (MEK)
family protein array can be made. Mitogen-activated protein kinases
(MAPKs) mediate many of the cellular effects of growth factors,
cytokines and stress stimuli. Their activation requires the
phosphorylation of a threonine and a tyrosine residue located in a
Thr-X-Tyr motif (where X is any amino acid) (Lawler S, et al.
Current Biology 1998, 8:1387-1390). This activation is carried out
by a family of enzymes known as MAP kinase (MKKs or MEKs). To date,
there are seven MKKs identified. These MKKs were found to activate
different MAPKs that involve in p44/p42 MAP kinase (Erk1/2)
pathway, SAPK/JNK pathway and p38 MAP kinase pathway, etc. MKK1 and
MKK2 activate p.sup.44/p42 MAP kinase in p44/p42 MAP kinase
(Erk1/2) pathway, MKK3, MKK4 and MKK6 activate p38 MAP kinase in
p38 MAP kinase pathway, MKK4 and MKK7 activate JNK in SAPK/JNK
pathway and MKK5 activates ERK5/BMK1 whose biological pathway is
await to be discovered. An MKK family protein array can consist of
most or all MKK family members. This protein array can allow
simultaneous identification of activated MKKs (in their
phosphorylated form) in a single assay. The activated MKKs indicate
activation of their corresponding signal pathways. Therefore, MKK
family protein array can serve as an efficient way for identifying
activated signal pathways.
[0182] Similarly, a MAP kinase family protein array can be made. In
mammalian systems, five distinguishable MAP kinase (MAPK) have been
identified. These MAPKs are involved in the extracellular
signal-regulated kinase 1 and 2 (ERK1/2) cascade, which
preferentially regulates cell growth and differentiation, as well
as the c-Jun N-terminal kinase (JNK) and p38 MAPK cascades, which
function mainly in stress responses like inflammation and
apoptosis. The last member ERK5 is activated by MKK5 and is thought
to transmit signal for proliferation. MAPK family protein array can
consist of most or all MAPK family members. This protein array
allows simultaneous identification of activated MAPKs (in their
phosphorylated form) in a single assay. The activated MAPKs
indicate activation of their corresponding signal pathways.
Therefore, MAPK family protein array can serve as an efficient way
for identifying activated signal pathways.
[0183] Groups of proteins whose modification signals activation of
a particular signal pathway can be studied and/or monitored using a
protein array, which can be called a pathwayfinder protein array.
The following two lists identify the pathway on the left and the
corresponding marker proteins on the right that would be captured
on the protein array and detected for their corresponding
modification.
1TABLE 1 Exemplary marker proteins for certain pathways Pathway
Marker protein P44/42 MAP kinase pathway phospho-MAPK 1 & 2
phospho MKK 1 & 2 P38 MAP kinase pathway phospho MKK 4 & 7
phospho-JNK phospho-c-Jun JNK pathway phospho p38 MAPK phospho MKK3
& 6 NFkB pathway phospho-ikB CREB pathway phospho-CREB P70 S6
kinase pathway phospho-p70 S6 kinase phospho-S6 PI-3 kinase/Akt
pathway phospho-Akt JAK/Stat pathway phospho-JAK or phospho-Stat
TGFb/Activin pathway phospho-Smad 2 & 3 BMP 2 & 4 pathway
phospho-Smad 1 & 5 BMP7 pathway phospho-Smad 5 NFAT pathway
dephosphorylated-NFAT1 Wnt pathway phospho-GSK3 P53 pathway
phospho-p53 Insulin signaling phospho-GSK3
[0184] A pathwayfinder protein array could contain antibodies
(capture molecules) against some of marker proteins listed above.
After reacting with a protein sample, the capture molecules would
capture the corresponding marker proteins, and the phosphorylation
state of these marker proteins would be detected by an appropriate
phosphorylation-specific antibody.
[0185] A protein array can be used to monitor enzymatic activities
of a group of proteins of interest. For example a group of specific
kinases can be studied for their activities, listed in Table 1, and
also elsewhere herein. Also a group of proteases can be studied for
their, activities, for example, a caspase family substrate array
can be made. Caspases are involved in apoptosis. There are a total
of 10 caspases identified to date. Caspase family substrate array
can allow simultaneous analyzing of several or all caspases for
their activities in a single assay. In caspase family substrate
array, the substrate for each caspase is immobilized on a solid
surface. These substrates are usually in colormetric or
fluorometric format and can be detected after the cleavage. For
example, a fluorescent or a colormetric dye is attached to the
substrate molecules such as DEVD as Caspase-3 substrate or IETD for
Caspase-8 substrate. Upon cleavage of the substrate by caspase, the
fluorescent or color signal is decreased proportionally to the
activity of corresponding caspase. Alternatively, two dye molecules
that quench their fluorescent signal can be attached to a single
substrate. After the cleavage by the caspase resulting a loss of a
fluorescent dye, a fluorescent signal is generated from the
residual substrate.
[0186] Various activation states of a protein can be monitored
using a protein array. For example, an Rb phosphorylation site
protein array can identify important activation states of
retinoblastoma (Rb). The retinoblastoma tumor suppressor, Rb,
regulates cell proliferation by controlling progression through the
restriction point within the GI phase of the cell cycle. Rb has
three functionally distinct binding domains and interacts with
critical regulatory proteins including the E2F family of
transcription factors, c-abl tyrosine kinase and proteins with a
conserved LXCXE motif. Cell cycle-dependent phosphorylation by cdks
controls Rb activity by preventing binding to these regulatory
targets. Rb can be phosphorylated at a multiplicity of sites and
differential phosphorylation has been shown to modulate Rb function
both in vitro and in vivo. Rb phosphorylation site protein array
contains a group of phospho-Rb antibodies, each against a specific
form of phospho-Rb. These antibodies can include anti-phospho-Rb
(Ser795), anti-phospho-Rb (Ser249/252), anti-phospho-Rb (Thr373),
anti-phospho-Rb (Ser780), anti-phospho-Rb (Ser8O7/811). After
capturing various form of Rb on the protein array, a Rb antibody
recognizing both unphosphorylated or phosphorylated Rb is used to
determined the amount of Rb at each conformation.
[0187] Kits of the invention are designed to detect protein
modification in a biologically active sample of proteins. The kits
comprise a solid support of 2 or more capture molecules immobilized
on the solid support, each of which can specifically bind a target
protein-that is capable of a subject protein modification; a
detection molecule specific for the subject protein modification in
order to detect whether or not a captured target protein comprises
the subject protein modification, wherein a target protein
comprising the subject protein modification imparts information
about biological activity present in the sample; and instructions
for use of the kit. The instructions may follow many of the
guidelines set forth above for practicing the method of the
invention. The kit components are as described above for the solid
supports and detection molecules. Other reagents, tools and/or
buffers may also be included in the kits. The kits may also
comprise containers for the kit contents.
[0188] The invention comprises also a composition comprising a
solid support comprising 2 or more capture molecules immobilized on
the support each of which can specifically bind a target protein
that is capable of protein modification; wherein target proteins
that specifically bind the capture molecules will form binding
pairs on the support; and a bound target protein on the support can
be detected with a detection molecule specific for protein
modification; further wherein if a target protein comprises protein
modification, information about biological activity present in a
sample that comprised the target proteins is imparted. The solid
supports are constructed and prepared essentially as described
above and elsewhere herein. The subject protein modification on a
solid support can comprises a modification selected from the group
consisting of phosphorylation, acetylation, methylation,
ADP-ribosylation, addition of a polypeptide side chain, addition of
a hydrophobic group, and addition of a carbohydrate. The subject
protein modification on the solid support can be a phosphorylation
and the phosphorylation can comprises tyrosine, serine or threonine
phosphorylation. The subject protein modification can be is
addition of a polypeptide side chain, and the polypeptide side
chain can be, for example, ubiquitin. The subject protein
modification can be addition of a hydophobic group and the
hydrophobic group can comprises a hydrophobic moiety selected from
the group consisting of a fatty acid, an isoprenoid, and a
glycosyl-phosphatidyl inositol anchor. The fatty acid can be
myristate or palmitate. The isoprenoid can be farnesyl or
genranylgenranyl. The carbohydrate addition can comprise a
glycosylation. The capture molecules on the solid support can be
antibodies. The detection molecules can be antibodies or lectins.
The solid support can comprise any types-of solid support described
above. The biological activity present in the sample can comprise
any biological activity that is implicated by a protein
modification, including any described herein.
[0189] Applying the principles of the basic method of the
invention, the invention also provides a method of identifying and
characterizing a changed condition. The changed condition is
capable of manifestation e.g. by protein modification or enzymatic
activity. The method is practiced by contacting two solid supports
each comprising 2 or more capture molecules immobilized on the
support with a first and second sample comprising target proteins;
wherein each solid support comprises an identical amount and
pattern of capture molecules and each capture molecule on each
solid support can specifically bind a target protein that is
capable of a subject protein modification; and further wherein the
first sample represents an unchanged condition and the second
sample represents a changed condition; contacting the first and
second solid support with detection molecules capable of detecting
modulated target proteins bound to capture molecules on the solid
support; and comparing the detected modifications on the first and
second solid supports to identify and characterize the changed
condition. The changed condition can be a change in any condition
biologically possible provided the change in the condition may be
detected by the presence or absence of a protein modification or
enzymatic activity present in the test protein sample in which the
condition may have occurred or is occurring. Thus, the changed
condition may comprise, for example, a condition selected from the
group consisting of a disease, a drug treatment, a chemical
treatment, a test drug effect, a physical change, a biological
change, a developmental stage, a disease stage, and a disease
progression.
[0190] Exemplary protein arrays may be described, but the following
exemplary arrays and those depicted in the subsequent tables are
not limiting of the invention, and by no means provide an
exhaustive review of all types of protein arrays or solid
substrates or subject protein modifications or subject enzymatic
activity possibly useful along the principles of the invention. A
protein array may comprising immobilized capture molecules on a
solid support capable of specifically binding certain proteins,
wherein the capture molecules are specific for 2 or more proteins
selected from the group consisting of Rac, MEKK3, MEK4, MEK7, JNK1,
JNK2, c-jun, Elk-1, Jun D, and ATF-4. A protein array may comprise
immobilized capture molecules on a solid support capable of
specifically binding certain proteins, wherein the capture
molecules are specific for a 2 or more of proteins consisting of
mitogenic pathway group comprising phosphorylation of any of
p.sup.44/42 MAP Kinase (Thr202/Tyr204) and MEK1/2 (Ser217/221), a
stress pathway group comprising phosphorylation of any of SAPK/JNK
(Thr183/Tyr185), p38 MAP kinase (Thr180/Tyr182), MKK3/MKK6
(Ser189/207), and SEK1/MKK4 (Thr223), a cell survival pathway group
comprising phosphorylation of any of Akt (Ser473), Bad (Serll2/136)
and p70 S6Kinase (Ser411, Thr421/Ser424), activation of NFkB signal
pathway comprising phosphorylation of ikB, activation of
mitogenic/differentiation signal pathway comprising phosphorylation
of any of ELK1, CREB, Etsl, Ets2, CBP, PEA3, p90.sup.rsk and CEBP,
activation of cytoskeletal organization signal pathway comprising
phosphorylation of any of c-Jun, Elk1, ATF2, c-myc, SAPla and PEA3,
and apoptosis/stress signal pathway comprising phosphorylation of
any of ATF1, ATF2, Elk1, Max, CHOP, CREB, SAP1a and MAPKAPK-2. A
protein array may comprising immobilized capture molecules on a
solid support capable of specifically binding certain proteins,
wherein the capture molecules are specific for 2 or more proteins
selected from the group consisting of phosphorylated proteins of
the ErbB family receptors comprising EGFR, ErbB-2, ErbB-3 and
ErbB-4. A protein array may comprise immobilized capture molecules
on a solid support capable of specifically binding certain
proteins, wherein the capture molecules are specific for 2 or more
phosphorylated proteins selected from the group consisting of EGF
receptor, PDGF receptor, SOS, Src, and p44/42 MAP Kinase. A protein
array may comprise immobilized capture molecules on a solid support
capable of specifically binding certain proteins, wherein the
capture molecules are specific for 2 or more phosphorylated
proteins selected from the group consisting of p44/42 MAP Kinase,
MEK1/2, SAPK/JNK, p38 MAP kinase, MKK3/MKK6, SEK1/MKK4, Akt, Bad
and p70 S6 Kinase. A protein array may comprise immobilized capture
molecules on a solid support capable of specifically binding
certain proteins, wherein the capture molecules are specific for 2
or more proteins selected from the group consisting of c-Jun,
ATF-2, Jun B, p53, b-catenin and cyclin D1. A protein array may
comprise immobilized capture molecules on a solid support capable
of specifically binding certain proteins, wherein the capture
molecules are specific for 2 or more genranylgenranylated proteins
selected from the group consisting of Rap1A/Krev1, Rac, Ral and
Rho. A protein array comprising immobilized capture molecules on a
solid support capable of specifically binding certain proteins,
wherein the capture molecules are specific for 2 or more
farnesylated proteins selected from the group consisting of H-Tas,
N-Ras, K-Ras, Lamins A, Lamin B, transductin T subunit, and
Rhodopsin kinase. A protein array comprising immobilized capture
molecules on a solid support capable of specifically binding
proteins active in a function selected from the group consisting of
mitogenesis, insulin activation or deactivation, apoptosis, cell
survival, stress signaling, geranylation, farnesylation, tyrosine
phosphorylation, serine phosphorylation, threonine phosphorylation,
kinase activity, NFKB activation, JAK/STAT signaling,
ubiquitination, proteins having lipid moieties, protein kinase C
signaling, cell adhesion, cytoskeletal organization, and receptor
signaling.
[0191] Exemplary enzyme substrate arrays can include, for example,
the peptide sequence EAIYAAPFAKKK as a peptide substrate for Abl
protein tyrosine kinase. Another substrate: KRQQSFDLF can be a
peptide substrate for calmodulin-dependent protein kinase. Protein
substrates can include, for example, ATF-2 as a substrate for p38
kinase and SAPK/JNK. c-Jun can be a substrate for SAPK/JNK kinase.
Elk1 can be a protein substrate for MAPK/ERK and SAPK/JNK. Inactive
p.sup.42 MAP kinase can be a protein substrate for MEK1 and/or MEK2
kinases. MBP can be a protein substrate for c-Raf kinase. With some
of these protein substrates, the cite of activity (e.g. the peptide
sequence) may suffice as a substrate for detecting the kinase
activity. Thus, for example, a protein array for kinase enzymatic
activity could include, e.g. ATF-2, c-Jun, Elk1, inactive p42 MAP
kinase and MBP as substrates for the respective above identified
kinase enzymes.
2TABLE 2 Exemplary Target Proteins for Various Protein Arrays Far-
gen- p38 MAP p44/p21 Kinases nesyla- genra- JNK kinase Ubiquit- MAP
(enzynme tion nylation stress Stress ination kinase AKT substrate)
H- Rap1A MKK4 p38 c-Jun p44 MAP Akt ATF-2 Tas MAPK kinase Ser473
substrate Thr180 Thr202 Thr308 for p38 Tyr182 Tyr204 kinase and
SAPK/JNK N- Krev1 MKK7 MKK3 ATF-2 p42 MAP Bad c-Jun Ras Ser189
kinase Ser112 substrate Ser207 Thr202 Ser136 for Tyr204 SAPK/JNK
kinase K- Rac JNK1 MKK6 Jun B MEK1 p70- Elk1 Ras Ser189 Ser217 S6
substrate Ser207 Ser221 kinase for Ser411 MAPK/ERK Thr421 and
SAPK/JNK Ser424 Lamins Ral JNK2 SEK1 p53 MEK2 GSK3 inactive A MKK4
Ser217 p42 MAP Thr223 Ser221 kinase substrate for MEK1/2 kinases
Lamin Rho c-jun ATF1 b- ELK1 MBP B catenin Ser383 substrate Ser389
for c-Raf kinase trans- ELK1 ATF2 cyclin Stat3 ductin D1 Ser727
.tau. subu- nit Rho- ATF2 ELK1 Etsl dopsin Ser383/389 kinase c-myc
MAX SAP1a Ser62 SAP1a CHOP CREB PEA3 CREB CBP Sen133 SAP1a PEA3
c-myc CEBP Ser62
[0192] The following example is included for illustrative purposes
only and is not intended to limit the scope of the invention.
F. EXAMPLES
[0193] 1. Preparation of a Membrane Protein Array
[0194] A nitrocellulose membrane (2.times.4 CM Protran BA85,
S&S) in soaked in phosphate buffered saline (PBS). Antibodies
specific for the target proteins of interest (listed below) are
diluted 1:100 in PBS. The diluted antibodies are spotted in 25 ul
aliquots onto a nitrocellulose membrane through a well created by a
dot blot apparatus according to the position indicated below. The
membrane is washed in PBS, and kept in PBS containing 0. 1%
thimerosal. The immobilized proteins are listed as follows (see
FIG. 2):
3 A1: c-erB2 (Neomarker, located at Union City, CA) A2: c-erB3
(Santa Cruz Biotechnology, located at Santa Cruz, CA) A3: c-erB4
(Neomarker) A4: actin (Sigma, located at St. Louis, MO) B1: EGFR
(Santa Cruz) B2: FGFR (UBI (Upstate Biotechnology) located at Lake
Placid, NY) B3: IGFR (Santa Cruz) B4: c-fes (Oncogen Science,
located at Cambridge, MA)
[0195] 2. Sample Preparation
[0196] Cell lines NIH3T3, NIH3T3/EGFR and NIH3T3/c-erB2 were
cultured in DMEM containing 5% FBS to 70% confluence. Cells were
harvested and sonicated in lysis buffer (50 mM Tris.Cl, pH 7.4, 150
mM NaCl, 1 mM EGTA, 1 mM Na3VO4, 100 nM okadaic acid, 1 mM PMSF, 1
ug/ml aprotinin, leupeptin and pepstatin, and 1% NP40). Cell lysate
was centrifuged at 14,000 rpm for 5 min, and supernatant was
diluted to protein concentration of 1 mg/ml.
[0197] 3. Hybridization with Protein Tyrosine Kinase (PTK) Membrane
Protein Array
[0198] PTK protein array membranes were incubated with
DetectorBlock solution (KPL, Gaitheburgh, Md.) for 1 hr. Rinse
membrane with TBST (50 mM Tris.Cl, pH 7.0, 150 mM NaCl, 0.1% Tween
20). 100 ul cell lysate was dropped on plastic wrap, then the
membrane PTK was placed down facing the cell lysate. The membrane
was covered with plastic wrap to prevent evaporation, and kept at
room temperature for 1 hr. The membrane was washed with TBST for 10
min 3 times. The membrane was incubated with 1:1000 diluted
anti-phosphotyrosine-peroxidase (anti-P-Try-POD) (Boehringer
Mannheim) in TBST for 45 min. The membrane was washed with TBST for
10 min 3 times. The membrane was incubated with ECL reagent
(Amersham) for 1 min, then wrapped with plastic wrap. The membrane
was exposed to X-ray film. The results indicated a positive at
position A1 on the membrane that was hybridized with cell lysate
prepared from c-erb2 transfected NIH3T3 cells, indicating that a
tyrosine phosphorylated c-erb2 target protein was in that cell
lysate.
[0199] (1) membrane 1 was hybridized with cell lysate prepared from
NIH3T3 cells;
[0200] (2) membrane was hybridized with cell lysate prepared from
EGFRtransfected NIH3T3 cells;
[0201] (3) membrane was hybridized with cell lysate prepared from
c-erb2 transfected NIH3T3 cells. Membrane (3) indicated a signal at
a location on the upper left-hand corner of the membrane at
position A1.
[0202] 4. Testing ErbB Family Receptor Protein Arrays
[0203] To determine the optimal conditions for a protein array for
the ErbB family of receptors (also called the EGF family of
receptors), each ErbB receptor can be tested to establish the
conditions for the entire array in order to optimize the
specificity for the ErbB protein array. A solid support is spotted
with capture molecules (antibodies) specific for the members of the
ErbB receptor family (e.g. EFGR, ErbB-2, ErbB-3, ErbB-4). IMDA-468
cell line (Kassis J, et al., Clin Cancer Res 1999 Aug;5(8):2251-60;
Reddy KB, et al., Int J Oncol 1999 Aug;15(2):301-6) which expresses
both EGFR and ErbB-3 receptor is treated with EGF to stimulate and
derive tyrosine-phosphorylated EGFR while ErbB-3 remains
unphosphorylated. To evaluate the specificity the protein array
assay for EGFR, the cell lysate from MDA-468 cells is incubated
with the ErbB protein array. The specific detection of EGFR using
the protein array assay should ensure no signals obtained from the
ErbB-2 receptor, ErbB-3 and ErbB-4 spots and a strong signal from
EGFR spot.
[0204] A 32D cell line (Ruggiero, M et al., 1991, FEBS, 291:203)
transfected with ErbB-2 is stimulated by heregulin to derive
tyrosine-phosphorylated ErbB-2. Specific detection of ErbB-2 using
the protein array assay ensures no signals obtained from the EGFR
receptor, ErbB-3 and ErbB-4 spots and a strong signal from ErbB-2
spot.
[0205] A 32D cell line transfected with ErbB-3 is stimulated by
heregulin to derive tyrosine-phosphorylated ErbB-3. Specific
detection for ErbB-3 using the protein array assay ensures no
signals obtained from the EGFR receptor, ErbB-2 and ErbB-4 spots
and a strong signal from ErbB-3 spot.
[0206] A 32D cell line transfected with ErbB-4 is stimulated by
heregulin to derive tyrosine-phosphorylated ErbB-4. Specific
detection for ErbB-4 using the protein array assay ensures no
signals obtained from the EGFR receptor, ErbB-2 and ErbB-3 spots
and a strong signal from ErbB-4 spot.
[0207] The dosage response of tyrosine phosphorylation of ErbB
family receptors can be detected by the protein array for that
family and compared with dosage responses detected by Westernblot
analysis. MDA-468 cells are stimulated with EGF at 3000, 1000, 333,
111, 37, 12, 4 or 0 pM separately. An aliquot of the lysate from
each sample is prepared and incubated with the ErbB protein array
to determine the level of tyrosine phosphorylation. An equal
aliquot of the lysate is immunoprecipitated with EGRF antibody
followed by Westernblot to determine for the level of tyrosine
phosphorylation on EGFR. This process is repeated for each other
target protein in the array (ErbB-2, 3, & 4) using the 32D cell
line, stimulation with heregulin at 3000, 1000, 333, 111, 37, 12, 4
or 0 pM, and in each case an aliquot of the lysate from each sample
is prepared and incubated with the ErbB protein array to determine
a level of tyrosine phosphorylation, and these results are compared
and correlated with results from both Westerblot analysis and the
proposed protein array analysis.
[0208] A screen of 12 tumor cell lines is established for ErbB
receptor family activation using an ErbB protein array to compare
the cell lines for the presence of the tyrosine phosphorylated
receptors. Twelve well-characterized tumor cell lines are used
(MDA-453, BT-474, MDA-361, N87, MCF-7, SKBr3, MDA-468, A431,
MDA-231, LCC6, SKOv3 and MCF10A (available at the ATCC; Yang D et
al., 1998, Clinical Cancer Research 4:993-1004). These cells are
stimulated with EGF and heregulin. The lysate from each of these
cell lines is used to incubate with the ErbB protein array to
determine the individual level of tyrosine phosphorylation of four
ErbB receptors. The level of tyrosine phosphorylation of these ErbB
receptors is also determined by Westernblot analysis. These
comparative data are then correlated.
[0209] A cell line from group of 12 cancer cell lines tested above
is selected that has the highest amount of activation of the ErbB
family receptors in the ErbB protein array assay. This cell line is
selected for screening for ErbB receptor activation inhibitors.
Multiple different potential inhibitors of ErbB receptor activation
are selected for screening, and the cell line is administered these
test drugs. The cell lysate contacts an ErbB receptor array and a
tyrosine phosphorylation antibody detects the identity and levels
of phosphorylated target proteins that are captured by the capture
molecules on the solid support. Candidate molecules for inhibiting
ErbB receptor activation are then selected. Some candidates may
appear to work most effectively on some but not all ErbB receptors,
and thus cocktails of the test drugs may also be tested.
[0210] Since modifications will be apparent to those of skill in
this art, it is intended that this invention be limited only by the
scope of the appended claims.
* * * * *